Radioimmunoconjugates and checkpoint inhibitor combination therapy

ABSTRACT

Combination therapies comprising administering radioimmunoconjugates and one or more checkpoint inhibitors.

RELATED APPLICATION

The present application claims priority to U.S. Provisional PatentApplication No. 62/774,847 filed Dec. 3, 2018, the entire contents ofwhich are hereby incorporated by reference for all purposes.

BACKGROUND

Cancer cells employ a variety of mechanisms to escape immunesurveillance, including suppression of T cell activation.

The mammalian immune system relies on checkpoint molecules todistinguish normal cells from foreign cells. Checkpoint molecules,expressed on certain immune cells, need to be activated or inactivatedto start an immune response. Inhibition of checkpoint proteins resultsin increased activation of the immune system.

Checkpoint inhibition has been explored as a method of immunotherapy forcancer. Inhibiting checkpoint proteins may activate T-cells and allowthem to attack cancer cells. However, checkpoint inhibition can allowthe immune system to attack some normal cells in the body, which canlead to serious side effects. In addition, some checkpoint inhibitorshave exhibited only modest efficacy in the clinic. There remains a needfor improved treatments of cancer. In particular, there is a need forincreases in efficacy, which do not enhance toxicity in the patient.

SUMMARY

The present disclosure encompasses the insight that combining inhibitionof checkpoint proteins with a therapy that targets damage to cancercells may provide a less toxic therapy with improved efficacy.Radioactive decay can cause direct physical damage (such as single ordouble-stranded DNA breaks) or indirect damage (such as by-stander orcrossfire effects) to the biomolecules that constitute a cell. Thepresent disclosure combines radioimmunoconjugates targeted to cancercells with checkpoint inhibition to induce or improve an immune responseto a tumor. In some embodiments, disclosed combination therapiesameliorate or treat cancer.

In one aspect, provided are methods of inducing an immune response to atumor in a mammal, said methods comprising: (i) administering to themammal a radioimmunoconjugate, wherein the mammal has received or isreceiving one or more checkpoint inhibitors; (ii) administering to themammal one or more checkpoint inhibitors, wherein the mammal hasreceived or is receiving a radioimmunoconjugate; or (iii) administeringthe mammal one or more checkpoint inhibitors at the same time asadministering the mammal a radioimmunoconjugate.

In some embodiments, said method comprises administering to a mammal oneor more checkpoint inhibitors, wherein the mammal has received or isreceiving a radioimmunoconjugate. In some embodiments, the one or morecheckpoint inhibitors is administered in a lower effective dose. In someembodiments, the radioimmunoconjugate is administered in a lowereffective dose. In some embodiments, both the one or more checkpointinhibitors and the radioimmunoconjugate are administered in lowereffective doses.

In some embodiments, the radioimmunoconjugate comprises (i) a targetingmoiety, (ii) a linker, and (iii) a chelating moiety or a metal complexof a chelating moiety.

In some embodiments, the targeting moiety is capable of binding to atumor-associated antigen.

In some embodiments, the tumor-associated antigen is a tumor-specificantigen.

In some embodiments, the targeting moiety is an antibody or anantigen-binding fragment thereof.

In some embodiments, the antibody or antigen-binding fragment thereof isan IGF-1R antibody or an antigen-binding fragment thereof.

In some embodiments, the antibody or antigen-binding fragment thereof isan endosialin (TEM-1) antibody or an antigen-binding fragment thereof.

In some embodiments, the radioimmunoconjugate comprises a metal complexof a chelating moiety. In some embodiments, the metal complex comprisesa radionuclide. In some embodiments, the radionuclide is an alphaemitter, e.g., an alpha emitter selected from the group consisting ofAstatine-211 (²¹¹At), Bismuth-212 (²¹²Bi), Bismuth-213 (²¹³Bi),Actinium-225 (²²⁵Ac), Radium-223 (²²³Ra), Lead-212 (²¹²Pb), Thorium-227(²²⁷Th), and Terbium-149 (¹⁴⁹Tb). In some embodiments, the radionuclideis ²²⁵Ac.

In some embodiments, the radioimmunoconjugate comprises the followingstructure:

wherein B is the targeting moiety.

In some embodiments, the one or more checkpoint inhibitors comprise aPD-1 inhibitor. In some embodiments, the PD-1 inhibitor is an antibody.

In some embodiments, the one or more checkpoint inhibitors comprise aCTLA-4 inhibitor. In some embodiments, the CTLA-4 inhibitor is anantibody.

In some embodiments, the one or more checkpoint inhibitors comprisesboth a PD-1 inhibitor and a CTLA-4 inhibitor.

In some embodiments, the mammal is a human.

In some embodiments, the mammal is diagnosed with cancer.

In some embodiments, the cancer is selected from the group comprising:breast cancer, non-small cell lung cancer, small cell lung cancer,pancreatic cancer, head and neck cancer, prostate cancer, colorectalcancer, sarcoma, adrenocortical carcinoma, neuroendocrine cancer,Ewing's Sarcoma, multiple myeloma, or acute myeloid leukemia.

In some embodiments, the mammal has at least one solid tumor.

In some embodiments, said administering results in a therapeutic effect.In some embodiments, said therapeutic effect comprises a decrease intumor volume, a stable tumor volume, or a reduced rate of increase intumor volume. In some embodiments, said therapeutic effect comprises adecreased incidence of recurrence or metastasis.

In some embodiments, the targeting moiety is capable of binding to atumor-associated antigen, and said therapeutic effect comprises anincrease in T cells specific for the tumor-associated antigen. In someembodiments, said increase in T cells occurs in the tumor. In someembodiments, said increase in the T cells in the tumor is relative to Tcells in the spleen.

In some embodiments, said administering results in at least 15% of thetotal T cell population in a sample from the mammal being specific forthe tumor-associated antigen.

In some embodiments, said administering results in at least 20% of thetotal T cell population in a sample from the mammal being specific forthe tumor-associated antigen.

In some embodiments, said administering results in at least 25% of thetotal T cell population in a sample from the mammal being specific forthe tumor-associated antigen.

In some embodiments, said administering results in at least 30% of thetotal T cell population in a sample from the mammal being specific forthe tumor-associated antigen.

In some embodiments, wherein said administering results in at least 35%of the total T cell population in a sample from the mammal beingspecific for the tumor-associated antigen.

In some embodiments, said administering results in at least 40% of thetotal T cell population in a sample from the mammal being specific forthe tumor-associated antigen.

In some embodiments, said administering results in at least 45% of thetotal T cell population in a sample from the mammal being specific forthe tumor-associated antigen.

In some embodiments, said administering results in at least 50% of thetotal T cell population in a sample from the mammal being specific forthe tumor-associated antigen.

In some embodiments, said administering results in at least 55% of thetotal T cell population in a sample from the mammal being specific forthe tumor-associated antigen.

In some embodiments, said administering results in at least 60% of thetotal T cell population in a sample from the mammal being specific forthe tumor-associated antigen.

In some embodiments, said administering results in at least 65% of thetotal T cell population in a sample from the mammal being specific forthe tumor-associated antigen.

In some embodiments, said administering results in at least 70% of thetotal T cell population in a sample from the mammal being specific forthe tumor-associated antigen.

In some embodiments, the sample is a tumor sample.

Definitions Chemical Terms

The term “acyl,” as used herein, represents a hydrogen or an alkyl group(e.g., a haloalkyl group), as defined herein, that is attached to theparent molecular group through a carbonyl group, as defined herein, andis exemplified by formyl (i.e., a carboxyaldehyde group), acetyl,trifluoroacetyl, propionyl, butanoyl and the like. Exemplaryunsubstituted acyl groups include from 1 to 7, from 1 to 11, or from 1to 21 carbons. In some embodiments, the alkyl group is furthersubstituted with 1, 2, 3, or 4 substituents as described herein.

The term “alkyl,” as used herein, is inclusive of both straight chainand branched chain saturated groups from 1 to 20 carbons (e.g., from 1to 10 or from 1 to 6), unless otherwise specified. Alkyl groups areexemplified by methyl, ethyl, n- and iso-propyl, n-, sec-, iso- andtert-butyl, neopentyl, and the like, and may be optionally substitutedwith one, two, three, or, in the case of alkyl groups of two carbons ormore, four substituents independently selected from the group consistingof: (1) C₁₋₆ alkoxy; (2) C₁₋₆ alkylsulfinyl; (3) amino, as definedherein (e.g., unsubstituted amino (i.e., —NH₂) or a substituted amino(i.e., —N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀aryl-C₁₋₆ alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8)hydroxy, optionally substituted with an O-protecting group; (9) nitro;(10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇ spirocyclyl; (12)thioalkoxy; (13) thiol; (14) —CO₂R^(A′) optionally substituted with anO-protecting group and where R^(A′) is selected from the groupconsisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀ alkenyl(e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆ alk-C₆₋₁₀aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N11), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′) whereeach of R^(E′) and R^(F′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′) where R^(G′) is selected from the groupconsisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀ alkenyl(e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆ alk-C₆₋₁₀aryl, (f amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R^(I′), wherein R^(H′) is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I′) is selected from thegroup consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein. For example, the alkylene group of aC₁-alkaryl can be further substituted with an oxo group to afford therespective aryloyl substituent.

The term “alkylene” and the prefix “alk-,” as used herein, represent asaturated divalent hydrocarbon group derived from a straight or branchedchain saturated hydrocarbon by the removal of two hydrogen atoms, and isexemplified by methylene, ethylene, isopropylene, and the like. The term“C_(x-y) alkylene” and the prefix “C_(x-y) alk-” represent alkylenegroups having between x and y carbons. Exemplary values for x are 1, 2,3, 4, 5, and 6, and exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 14, 16, 18, or 20 (e.g., C₁₋₆, C₁₋₁₀, C₂₋₂₀, C₂₋₆, C₂₋₁₀, orC₂₋₂₀ alkylene). In some embodiments, the alkylene can be furthersubstituted with 1, 2, 3, or 4 substituent groups as defined herein foran alkyl group.

The term “alkenyl,” as used herein, represents monovalent straight orbranched chain groups of, unless otherwise specified, from 2 to 20carbons (e.g., from 2 to 6 or from 2 to 10 carbons) containing one ormore carbon-carbon double bonds and is exemplified by ethenyl,1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, andthe like. Alkenyls include both cis and trans isomers. Alkenyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from amino, aryl, cycloalkyl, orheterocyclyl (e.g., heteroaryl), as defined herein, or any of theexemplary alkyl substituent groups described herein.

The term “alkynyl,” as used herein, represents monovalent straight orbranched chain groups from 2 to 20 carbon atoms (e.g., from 2 to 4, from2 to 6, or from 2 to 10 carbons) containing a carbon-carbon triple bondand is exemplified by ethynyl, 1-propynyl, and the like. Alkynyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from aryl, cycloalkyl, or heterocyclyl(e.g., heteroaryl), as defined herein, or any of the exemplary alkylsubstituent groups described herein.

The term “amino,” as used herein, represents —N(R^(N1))₂, wherein eachR^(N1) is, independently, H, OH, NO₂, N(R^(N2))₂, SO₂OR^(N2), SO₂R^(N2),SOR^(N2), an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl,alkaryl, cycloalkyl, alkcycloalkyl, carboxyalkyl (e.g., optionallysubstituted with an O-protecting group, such as optionally substitutedarylalkoxycarbonyl groups or any described herein), sulfoalkyl, acyl(e.g., acetyl, trifluoroacetyl, or others described herein),alkoxycarbonylalkyl (e.g., optionally substituted with an O-protectinggroup, such as optionally substituted arylalkoxycarbonyl groups or anydescribed herein), heterocyclyl (e.g., heteroaryl), or alkheterocyclyl(e.g., alkheteroaryl), wherein each of these recited R^(N1) groups canbe optionally substituted, as defined herein for each group; or twoR^(N1) combine to form a heterocyclyl or an N-protecting group, andwherein each R^(N2) is, independently, H, alkyl, or aryl. Amino groupscan be unsubstituted amino (i.e., —NH₂) or substituted amino (i.e.,—N(R^(N1))₂) groups. In a preferred embodiment, amino is —NH₂ or—NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂, NR^(N22),SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, carboxyalkyl, sulfoalkyl, acyl(e.g., acetyl, trifluoroacetyl, or others described herein),alkoxycarbonylalkyl (e.g., t-butoxycarbonylalkyl) or aryl, and eachR^(N2) can be H, C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), or C₆₋₁₀ aryl.

The term “amino acid,” as described herein, refers to a molecule havinga side chain, an amino group, and an acid group (e.g., a carboxy groupof —CO₂H or a sulfo group of —SO₃H), wherein the amino acid is attachedto the parent molecular group by the side chain, amino group, or acidgroup (e.g., the side chain). In some embodiments, the amino acid isattached to the parent molecular group by a carbonyl group, where theside chain or amino group is attached to the carbonyl group. Exemplaryside chains include an optionally substituted alkyl, aryl, heterocyclyl,alkaryl, alkheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl.Exemplary amino acids include alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, histidine,hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline,ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine,taurine, threonine, tryptophan, tyrosine, and valine. Amino acid groupsmay be optionally substituted with one, two, three, or, in the case ofamino acid groups of two carbons or more, four substituentsindependently selected from the group consisting of: (1) C₁₋₆ alkoxy;(2) C₁₋₆ alkylsulfinyl; (3) amino, as defined herein (e.g.,unsubstituted amino (i.e., —NH₂) or a substituted amino (i.e.,—N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀ aryl-C₁₋₆alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8) hydroxy;(9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO₂R^(A′) where R^(A′)is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d)hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g)polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, whereins1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), eachof s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is Hor C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R_(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′) whereeach of R^(E′) and R^(F′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R^(I′), wherein R^(H′) is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I′) is selected from thegroup consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein sis aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein.

The term “aryl,” as used herein, represents a mono-, bicyclic, ormulticyclic carbocyclic ring system having one or two aromatic rings andis exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl,1,2,3,4-tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl,indanyl, indenyl, and the like, and may be optionally substituted with1, 2, 3, 4, or 5 substituents independently selected from the groupconsisting of: (1) C₁₋₇ acyl (e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl(e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl,halo-C₁₋₆ alkyl (e.g., perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆alkoxy, such as perfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo; (12) C₁₋₁₂heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂ heterocyclyl)oxy;(14) hydroxy; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g., C₁₋₆ thioalkoxy);(17) —(CH₂)_(q)CO₂R^(A′), where q is an integer from zero to four, andR^(A′) is selected from the group consisting of (a) C₁₋₆ alkyl, (b)C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (18)—(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to four andwhere R^(B′) and R^(C′) are independently selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q is an integer fromzero to four and where R^(D′) is selected from the group consisting of(a) alkyl, (b) C₆₋₁₀ aryl, and (c) alk-C₆₋₁₀ aryl; (20)—(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(F′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) C₂₋₂₀ alkenyl; and(27) C₂₋₂₀ alkynyl. In some embodiments, each of these groups can befurther substituted as described herein. For example, the alkylene groupof a C₁-alkaryl or a C₁-alkheterocyclylcan be further substituted withan oxo group to afford the respective aryloyl and (heterocyclyl)oylsubstituent group.

The term “arylalkyl,” as used herein, represents an aryl group, asdefined herein, attached to the parent molecular group through analkylene group, as defined herein. Exemplary unsubstituted arylalkylgroups are from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20carbons, such as C₁₋₆ alk-C₆₋₁₀ aryl, C₁₋₁₀ alk-C₆₋₁₀ aryl, or C₁₋₂₀alk-C₆₋₁₀ aryl). In some embodiments, the alkylene and the aryl each canbe further substituted with 1, 2, 3, or 4 substituent groups as definedherein for the respective groups. Other groups preceded by the prefix“alk-” are defined in the same manner, where “alk” refers to a C₁₋₆alkylene, unless otherwise noted, and the attached chemical structure isas defined herein.

The term “carbonyl,” as used herein, represents a C(O) group, which canalso be represented as C═O.

The term “carboxy,” as used herein, means —CO₂H.

The term “cyano,” as used herein, represents an —CN group.

The term “cycloalkyl,” as used herein represents a monovalent saturatedor unsaturated non-aromatic cyclic hydrocarbon group from three to eightcarbons, unless otherwise specified, and is exemplified by cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicycle heptyl, andthe like. When the cycloalkyl group includes one carbon-carbon doublebond or one carbon-carbon triple bond, the cycloalkyl group can bereferred to as a “cycloalkenyl” or “cycloalkynyl” group respectively.Exemplary cycloalkenyl and cycloalkynyl groups include cyclopentenyl,cyclohexenyl, cyclohexynyl, and the like. Cycloalkyl groups can beoptionally substituted with: (1) C₁₋₇ acyl (e.g., carboxyaldehyde); (2)C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl,(carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g., perfluoroalkyl),hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl);(3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such as perfluoroalkoxy); (4) C₁₋₆alkylsulfinyl; (5) C₁₋₁₀ aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8)azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo;(12) C₁₋₁₂ heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g.,C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′) where q is an integer fromzero to four, and R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl;(18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to fourand where R^(B′) and R^(C′) are independently selected from the groupconsisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and (d)C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q is an integerfrom zero to four and where R^(D′) is selected from the group consistingof (a) C₆₋₁₀ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀ aryl; (20)—(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(F′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) C₂₋₂₀alkenyl; and (28) C₂₋₂₀ alkynyl. In some embodiments, each of thesegroups can be further substituted as described herein. For example, thealkylene group of a C₁-alkaryl or a C₁-alkheterocyclyl can be furthersubstituted with an oxo group to afford the respective aryloyl and(heterocyclyl)oyl substituent group.

The term “diastereomer,” as used herein means stereoisomers that are notmirror images of one another and are non-superimposable on one another.

The term “enantiomer,” as used herein, means each individual opticallyactive form of a compound, having an optical purity or enantiomericexcess (as determined by methods standard in the art) of at least 80%(i.e., at least 90% of one enantiomer and at most 10% of the otherenantiomer), preferably at least 90% and more preferably at least 98%.

The term “halogen,” as used herein, represents a halogen selected frombromine, chlorine, iodine, or fluorine.

The term “heteroalkyl,” as used herein, refers to an alkyl group, asdefined herein, in which one or two of the constituent carbon atoms haveeach been replaced by nitrogen, oxygen, or sulfur. In some embodiments,the heteroalkyl group can be further substituted with 1, 2, 3, or 4substituent groups as described herein for alkyl groups. The terms“heteroalkenyl” and heteroalkynyl,” as used herein refer to alkenyl andalkynyl groups, as defined herein, respectively, in which one or two ofthe constituent carbon atoms have each been replaced by nitrogen,oxygen, or sulfur. In some embodiments, the heteroalkenyl andheteroalkynyl groups can be further substituted with 1, 2, 3, or 4substituent groups as described herein for alkyl groups.

The term “heteroaryl,” as used herein, represents that subset ofheterocyclyls, as defined herein, which are aromatic: i.e., they contain4n+2 pi electrons within the mono- or multicyclic ring system. Exemplaryunsubstituted heteroaryl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10,1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. In someembodiment, the heteroaryl is substituted with 1, 2, 3, or 4substituents groups as defined for a heterocyclyl group.

The term “heteroarylalkyl” refers to a heteroaryl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted heteroarylalkyl groupsare from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to12 carbons, such as C₁₋₆ alk-C₁₋₁₂ heteroaryl, C₁₋₁₀ alk-C₁₋₁₂heteroaryl, or C₁₋₂₀ alk-C₁₋₁₂ heteroaryl). In some embodiments, thealkylene and the heteroaryl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.Heteroarylalkyl groups are a subset of heterocyclylalkyl groups.

The term “heterocyclyl,” as used herein represents a 5-, 6- or7-membered ring, unless otherwise specified, containing one, two, three,or four heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur. The 5-membered ring has zero to two doublebonds, and the 6- and 7-membered rings have zero to three double bonds.Exemplary unsubstituted heterocyclyl groups are of 1 to 12 (e.g., 1 to11, 1 to 10, 1 to 9, 2 to 12, 2 to, 2 to 10, or 2 to 9) carbons. Theterm “heterocyclyl” also represents a heterocyclic compound having abridged multicyclic structure in which one or more carbons and/orheteroatoms bridges two non-adjacent members of a monocyclic ring, e.g.,a quinuclidinyl group. The term “heterocyclyl” includes bicyclic,tricyclic, and tetracyclic groups in which any of the above heterocyclicrings is fused to one, two, or three carbocyclic rings, e.g., an arylring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, acyclopentene ring, or another monocyclic heterocyclic ring, such asindolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl,benzothienyl and the like. Examples of fused heterocyclyls includetropanes and 1,2,3,5,8,8a-hexahydroindolizine. Heterocyclics includepyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl,pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl,piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl,pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl,morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl,quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl,phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl,benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl,triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl), purinyl,thiadiazolyl (e.g., 1,2,3-thiadiazolyl), tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl,dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl,dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl,isobenzofuranyl, benzothienyl, and the like, including dihydro andtetrahydro forms thereof, where one or more double bonds are reduced andreplaced with hydrogens. Still other exemplary heterocyclyls include:2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1H-imidazolyl;2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g.,2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl);2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g.,2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl);2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g.,2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl);4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl);2,6-dioxo-piperidinyl (e.g., 2,6-dioxo-3-ethyl-3-phenylpiperidinyl);1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (e.g.,2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl);1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl);1,6-dihydro-6-oxo-pyridazinyl (e.g.,1,6-dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl(e.g., 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl);2,3-dihydro-2-oxo-1H-indolyl (e.g.,3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and2,3-dihydro-2-oxo-3,3′-spiropropane-1H-indol-1-yl);1,3-dihydro-1-oxo-2H-iso-indolyl; 1,3-dihydro-1,3-dioxo-2H-iso-indolyl;1H-benzopyrazolyl (e.g., 1-(ethoxycarbonyl)-1H-benzopyrazolyl);2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g.,3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl);2,3-dihydro-2-oxo-benzoxazolyl (e.g.,5-chloro-2,3-dihydro-2-oxo-benzoxazolyl);2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl;1,4-benzodioxanyl; 1,3-benzodioxanyl;2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl;3,4-dihydro-4-oxo-3H-quinazolinyl (e.g.,2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl);1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g.,1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl);1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g.,1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl);1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g.,1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl);2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphth[1,8-c,d]isothiazolyl; and1,8-naphthylenedicarboxamido. Additional heterocyclics include3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanyl),tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl,thiepanyl, azocanyl, oxecanyl, and thiocanyl. Heterocyclic groups alsoinclude groups of the formula

where

E′ is selected from the group consisting of —N— and —CH—; F′ is selectedfrom the group consisting of —N═CH—, —NH—CH₂—, —NH—C(O)—, —NH—, —CH═N—,—CH₂—NH—, —C(O)—NH—, —CH═CH—, —CH₂—, —CH₂CH₂—, —CH₂O—, —OCH₂—, —O—, and—S—; and G′ is selected from the group consisting of —CH— and —N—. Anyof the heterocyclyl groups mentioned herein may be optionallysubstituted with one, two, three, four or five substituentsindependently selected from the group consisting of: (1) C₁₋₇ acyl(e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl,azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g.,perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such asperfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₁₋₁₀ aryl; (6) amino; (7)C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈cycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g., C₂₋₁₂ heteroaryl);(13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q isan integer from zero to four, and R^(A′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integerfrom zero to four and where R^(B′) and R^(C′) are independently selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q isan integer from zero to four and where R^(D′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀aryl; (20) —(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zeroto four and where each of R^(E′) and R^(F′) is, independently, selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₁₀ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23)C₃₋₈ cycloalkoxy; (24) arylalkoxy; (25) C₁₋₆ alk-C₁₋₁₂ heterocyclyl(e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) (C₁₋₁₂heterocyclyl)imino; (28) C₂₋₂₀ alkenyl; and (29) C₂₋₂₀ alkynyl. In someembodiments, each of these groups can be further substituted asdescribed herein. For example, the alkylene group of a C₁-alkaryl or aC₁-alkheterocyclyl can be further substituted with an oxo group toafford the respective aryloyl and (heterocyclyl)oyl substituent group.

The term “hydrocarbon,” as used herein, represents a group consistingonly of carbon and hydrogen atoms.

The term “hydroxyl,” as used herein, represents an —OH group. In someembodiments, the hydroxyl group can be substituted with 1, 2, 3, or 4substituent groups (e.g., 0-protecting groups) as defined herein for analkyl.

The term “isomer,” as used herein, means any tautomer, stereoisomer,enantiomer, or diastereomer of any compound. It is recognized that thecompounds can have one or more chiral centers and/or double bonds and,therefore, exist as stereoisomers, such as double-bond isomers (i.e.,geometric E/Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or(−)) or cis/trans isomers). Unless otherwise noted, chemical structuresdepicted herein encompass all of the corresponding stereoisomers, thatis, both the stereomerically pure form (e.g., geometrically pure,enantiomerically pure, or diastereomerically pure) and enantiomeric andstereoisomeric mixtures, e.g., racemates. Enantiomeric andstereoisomeric mixtures of compounds can typically be resolved intotheir component enantiomers or stereoisomers by well-known methods, suchas chiral-phase gas chromatography, chiral-phase high performance liquidchromatography, crystallizing the compound as a chiral salt complex, orcrystallizing the compound in a chiral solvent. Enantiomers andstereoisomers can also be obtained from stereomerically orenantiomerically pure intermediates, reagents, and catalysts bywell-known asymmetric synthetic methods.

The term “N-protected amino,” as used herein, refers to an amino group,as defined herein, to which is attached one or two N-protecting groups,as defined herein.

The term “N-protecting group,” as used herein, represents those groupsintended to protect an amino group against undesirable reactions duringsynthetic procedures. Commonly used N-protecting groups are disclosed inGreene, “Protective Groups in Organic Synthesis,” 3^(rd) Edition (JohnWiley & Sons, New York, 1999), which is incorporated herein byreference. N-protecting groups include acyl, aryloyl, or carbamyl groupssuch as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliariessuch as protected or unprotected D, L or D, L-amino acids such asalanine, leucine, phenylalanine, and the like; sulfonyl-containinggroups such as benzenesulfonyl, p-toluenesulfonyl, and the like;carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl,and the like and silyl groups, such as trimethylsilyl, and the like.Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc),and benzyloxycarbonyl (Cbz).

The term “O-protecting group,” as used herein, represents those groupsintended to protect an oxygen containing (e.g., phenol, hydroxyl, orcarbonyl) group against undesirable reactions during syntheticprocedures. Commonly used O-protecting groups are disclosed in Greene,“Protective Groups in Organic Synthesis,” 3d Edition (John Wiley & Sons,New York, 1999), which is incorporated herein by reference. ExemplaryO-protecting groups include acyl, aryloyl, or carbamyl groups, such asformyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl,2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl,o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl,4-bromobenzoyl, t-butyldimethylsilyl, tri-iso-propylsilyloxymethyl,4,4′-dimethoxytrityl, isobutyryl, phenoxyacetyl,4-isopropylpehenoxyacetyl, dimethylformamidino, and 4-nitrobenzoyl;alkylcarbonyl groups, such as acyl, acetyl, propionyl, pivaloyl, and thelike; optionally substituted arylcarbonyl groups, such as benzoyl; silylgroups, such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS),tri-iso-propylsilyloxymethyl (TOM), triisopropylsilyl (TIPS), and thelike; ether-forming groups with the hydroxyl, such methyl,methoxymethyl, tetrahydropyranyl, benzyl, p-methoxybenzyl, trityl, andthe like; alkoxycarbonyls, such as methoxycarbonyl, ethoxycarbonyl,isopropoxycarbonyl, n-isopropoxycarbonyl, n-butyloxycarbonyl,isobutyloxycarbonyl, sec-butyloxycarbonyl, t-butyloxycarbonyl,2-ethylhexyloxycarbonyl, cyclohexyloxycarbonyl, methyloxycarbonyl, andthe like; alkoxyalkoxycarbonyl groups, such as methoxymethoxycarbonyl,ethoxymethoxycarbonyl, 2-methoxyethoxycarbonyl, 2-ethoxyethoxycarbonyl,2-butoxyethoxycarbonyl, 2-methoxyethoxymethoxycarbonyl,allyloxycarbonyl, propargyloxycarbonyl, 2-butenoxycarbonyl,3-methyl-2-butenoxycarbonyl, and the like; haloalkoxycarbonyls, such as2-chloroethoxycarbonyl, 2-chloroethoxycarbonyl,2,2,2-trichloroethoxycarbonyl, and the like; optionally substitutedarylalkoxycarbonyl groups, such as benzyloxycarbonyl,p-methylbenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2,4-dinitrobenzyloxycarbonyl,3,5-dimethylbenzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-bromobenzyloxy-carbonyl, fluorenylmethyloxycarbonyl, and the like; andoptionally substituted aryloxycarbonyl groups, such as phenoxycarbonyl,p-nitrophenoxycarbonyl, o-nitrophenoxycarbonyl,2,4-dinitrophenoxycarbonyl, p-methyl-phenoxycarbonyl,m-methylphenoxycarbonyl, o-bromophenoxycarbonyl,3,5-dimethylphenoxycarbonyl, p-chlorophenoxycarbonyl,2-chloro-4-nitrophenoxy-carbonyl, and the like); substituted alkyl,aryl, and alkaryl ethers (e.g., trityl; methylthiomethyl; methoxymethyl;benzyloxymethyl; siloxymethyl; 2,2,2,-trichloroethoxymethyl;tetrahydropyranyl; tetrahydrofuranyl; ethoxyethyl;1-[2-(trimethylsilyl)ethoxy]ethyl; 2-trimethylsilylethyl; t-butyl ether;p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, benzyl, p-methoxybenzyl,and nitrobenzyl); silyl ethers (e.g., trimethylsilyl; triethylsilyl;triisopropylsilyl; dimethylisopropylsilyl; t-butyldimethylsilyl;t-butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; anddiphenymethylsilyl); carbonates (e.g., methyl, methoxymethyl,9-fluorenylmethyl; ethyl; 2,2,2-trichloroethyl; 2-(trimethylsilyl)ethyl;vinyl, allyl, nitrophenyl; benzyl; methoxybenzyl; 3,4-dimethoxybenzyl;and nitrobenzyl); carbonyl-protecting groups (e.g., acetal and ketalgroups, such as dimethyl acetal, 1,3-dioxolane, and the like; acylalgroups; and dithiane groups, such as 1,3-dithianes, 1,3-dithiolane, andthe like); carboxylic acid-protecting groups (e.g., ester groups, suchas methyl ester, benzyl ester, t-butyl ester, orthoesters, and the like;and oxazoline groups.

The term “oxo” as used herein, represents ═O.

The term “polyethylene glycol,” as used herein, represents an alkoxychain comprised of one or more monomer units, each monomer unitconsisting of —OCH₂CH₂—. Polyethyelene glycol (PEG) is also sometimesreferred to as polyethylene oxide (PEO) or polyoxyethylene (POE), andthese terms may be considered interchangeable for the purpose of thisdisclosure. For example, a polyethylene glycol may have the structure,—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)O—, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), and each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10). Polyethylene glycol mayalso be considered to include an amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl.

The term “stereoisomer,” as used herein, refers to all possibledifferent isomeric as well as conformational forms which a compound maypossess (e.g., a compound of any formula described herein), inparticular all possible stereochemically and conformationally isomericforms, all diastereomers, enantiomers and/or conformers of the basicmolecular structure. Some compounds may exist in different tautomericforms, all of the latter being included within the scope of the presentdisclosure.

The term “sulfonyl,” as used herein, represents an —S(O)₂— group.

The term “thiol,” as used herein represents an —SH group.

Other Terms

As used herein, the term “administered in combination,” “combinedadministration,” or “co-administered” means that two or more agents areadministered to a subject at the same time or within an interval suchthat there may be an overlap of an effect of each agent on the patient.Thus, two or more agents that are administered in combination need notbe administered together. In some embodiments, they are administeredwithin 90 days (e.g., within 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2,or 1 day(s)), within 28 days (e.g., with 14, 7, 6, 5, 4, 3, 2, or 1day(s), within 24 hours (e.g., 12, 6, 5, 4, 3, 2, or 1 hour(s), orwithin about 60, 30, 15, 10, 5, or 1 minute of one another. In someembodiments, the administrations of the agents are spaced sufficientlyclosely together such that a combinatorial effect is achieved.

As used herein, “administering” an agent to a subject includescontacting cells of said subject with the agent.

As used herein, “antibody” refers to a polypeptide whose amino acidsequence including immunoglobulins and fragments thereof whichspecifically bind to a designated antigen, or fragments thereof.Antibodies may be of any type (e.g., IgA, IgD, IgE, IgG, or IgM) orsubtype (e.g., IgA1, IgA2, IgG1, IgG2, IgG3, or IgG4). Those of ordinaryskill in the art will appreciate that a characteristic sequence orportion of an antibody may include amino acid sequences found in one ormore regions of an antibody (e.g., variable region, hypervariableregion, constant region, heavy chain, light chain, and combinationsthereof). Moreover, those of ordinary skill in the art will appreciatethat a characteristic sequence or portion of an antibody may include oneor more polypeptide chains and may include sequence elements found inthe same polypeptide chain or in different polypeptide chains.

As used herein, “antigen-binding fragment” refers to a portion of anantibody that retains the binding characteristics of the parentantibody.

The terms “bifunctional chelate” or “bifunctional conjugate” as usedinterchangeably herein, refer to a compound that contains a chelatinggroup or metal complex thereof, a linker group, and a therapeuticmoiety, targeting moiety, or cross-linking group.

The term “cancer” refers to any cancer caused by the proliferation ofmalignant neoplastic cells, such as tumors, neoplasms, carcinomas,sarcomas, leukemias, and lymphomas. A “solid tumor cancer” is a cancercomprising an abnormal mass of tissue, e.g., sarcomas, carcinomas, andlymphomas. A “hematological cancer” or “liquid cancer,” as usedinterchangeably herein, is a cancer present in a body fluid, e.g.,lymphomas and leukemias.

The term “checkpoint inhibitor,” also known as “immune checkpointinhibitor” or “ICI,” refers to an agent which blocks the action of animmune checkpoint protein, e.g., blocks such immune checkpoint proteinsfrom binding to their partner proteins.

The term “chelate” as used herein, refers to an organic compound orportion thereof that can be bonded to a central metal or radiometal atomat two or more points.

The term “conjugate,” as used herein, refers to a molecule that containsa chelating group or metal complex thereof, a linker group, and whichoptionally contains a therapeutic moiety, targeting moiety, orcross-linking group.

As used herein, the term “compound,” is meant to include allstereoisomers, geometric isomers, and tautomers of the structuresdepicted.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent disclosure that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically active starting materialsare known in the art, such as by resolution of racemic mixtures or bystereoselective synthesis. Many geometric isomers of olefins, C═N doublebonds, and the like can also be present in the compounds describedherein, and all such stable isomers are contemplated in the presentdisclosure. Cis and trans geometric isomers of the compounds of thepresent disclosure are described and may be isolated as a mixture ofisomers or as separated isomeric forms.

Compounds of the present disclosure also include tautomeric forms.Tautomeric forms result from the swapping of a single bond with anadjacent double bond and the concomitant migration of a proton.Tautomeric forms include prototropic tautomers which are isomericprotonation states having the same empirical formula and total charge.Examples prototropic tautomers include ketone-enol pairs, amide-imidicacid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-iminepairs, and annular forms where a proton can occupy two or more positionsof a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution.

At various places in the present specification, substituents ofcompounds of the present disclosure are disclosed in groups or inranges. It is specifically intended that the present disclosure includeeach and every individual subcombination of the members of such groupsand ranges. For example, the term “C₁₋₆ alkyl” is specifically intendedto individually disclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl,and C₆ alkyl. Herein a phrase of the form “optionally substituted X”(e.g., optionally substituted alkyl) is intended to be equivalent to “X,wherein X is optionally substituted” (e.g., “alkyl, wherein said alkylis optionally substituted”). It is not intended to mean that the feature“X” (e.g., alkyl) per se is optional.

The term “cross-linking group” as used herein refers to any reactivegroup that is able to join two or more molecules by a covalent bond. Insome embodiments, the cross-linking group is an amino-reactiveorthiol-reactive cross-linking group. In some embodiments, theamino-reactive orthiol-reactive cross-linking group comprises anactivated ester such as a hydroxysuccinimide ester,2,3,5,6-tetrafluorophenol ester, 4-nitrophenol ester or an imidate,anhydride, thiol, disulfide, maleimide, azide, alkyne, strained alkyne,strained alkene, halogen, sulfonate, haloacetyl, amine, hydrazide,diazirine, phosphine, tetrazine, isothiocyanate. In some embodiments,the cross-linking group may be glycine-glycine-glycine and/orleucine-proline-(any amino acid)-threonine-glycine, which are therecognition sequences for coupling targeting agents with the linkerusing a sortase-mediated coupling reaction. The person having ordinaryskill in the art will understand that the use of cross-linking groupsare not limited to the specific constructs disclosed herein, but rathermay include other known cross-linking groups.

As used herein, the terms “decrease,” “decreased,” “increase,”“increased,” or “reduction,” “reduced,” (e.g., in reference totherapeutic outcomes or effects) have meanings relative to a referencelevel. In some embodiments, the reference level is a level as determinedby the use of said method with a control in an experimental animal modelor clinical trial. In some embodiments, the reference level is a levelin the same subject before or at the beginning of treatment. In someembodiments, the reference level is the average level in a populationnot being treated by said method of treatment.

As used herein “detection agent” refers to a molecule or atom which isuseful in diagnosing a disease by locating the cells containing theantigen. Various methods of labeling polypeptides with detection agentsare known in the art. Examples of detection agents include, but are notlimited to, radioisotopes and radionuclides, dyes (such as with thebiotin-streptavidin complex), contrast agents, luminescent agents (e.g.,FITC, rhodamine, lanthanide phosphors, cyanine, and near IR dyes), andmagnetic agents, such as gadolinium chelates.

The term an “effective amount” of an agent (e.g., any of the foregoingconjugates), as used herein, is that amount sufficient to effectbeneficial or desired results, such as clinical results, and, as such,an “effective amount” depends upon the context in which it is beingapplied.

The term “immunoconjugate,” as used herein, refers to a conjugate thatincludes a targeting moiety, such as an antibody, nanobody, affibody, ora consensus sequence from Fibronectin type III domain. In someembodiments, the immunoconjugate comprises an average of at least 0.10conjugates per targeting moiety (e.g., an average of at least 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 4, 5, or 8 conjugates per targetingmoiety).

The term “lower effective dose,” when used as a term in conjunction withan agent (e.g., a therapeutic agent) refers to a dosage of the agentwhich is effective therapeutically in the combination therapies of theinvention and which is lower than the dose which has been determined tobe effective therapeutically when the agent is used as a monotherapy inreference experiments or by virtue of other therapeutic guidance.

The term “pharmaceutical composition,” as used herein, represents acomposition containing a compound described herein formulated with apharmaceutically acceptable excipient. In some embodiments, thepharmaceutical composition is manufactured or sold with the approval ofa governmental regulatory agency as part of a therapeutic regimen forthe treatment of disease in a mammal. Pharmaceutical compositions can beformulated, for example, for oral administration in unit dosage form(e.g., a tablet, capsule, caplet, gelcap, or syrup); for topicaladministration (e.g., as a cream, gel, lotion, or ointment); forintravenous administration (e.g., as a sterile solution free ofparticulate emboli and in a solvent system suitable for intravenoususe); or in any other formulation described herein.

A “pharmaceutically acceptable excipient,” as used herein, refers anyingredient other than the compounds described herein (for example, avehicle capable of suspending or dissolving the active compound) andhaving the properties of being nontoxic and non-inflammatory in apatient. Excipients may include, for example: antiadherents,antioxidants, binders, coatings, compression aids, disintegrants, dyes(colors), emollients, emulsifiers, fillers (diluents), film formers orcoatings, flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, radioprotectants, sorbents, suspending ordispersing agents, sweeteners, or waters of hydration. Exemplaryexcipients include, but are not limited to: ascorbic acid, histidine,phosphate buffer, butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc,titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

The term “pharmaceutically acceptable salt,” as use herein, representsthose salts of the compounds described here that are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof humans and animals without undue toxicity, irritation, or allergicresponse. Pharmaceutically acceptable salts are well known in the art.For example, pharmaceutically acceptable salts are described in: Bergeet al., J. Pharmaceutical Sciences 66:1-19, 1977 and in PharmaceuticalSalts: Properties, Selection, and Use, (Eds. P. H. Stahl and C. G.Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during thefinal isolation and purification of the compounds described herein orseparately by reacting the free base group with a suitable organic acid.

Compounds may have ionizable groups so as to be capable of preparationas pharmaceutically acceptable salts. These salts may be acid additionsalts involving inorganic or organic acids or the salts may, in the caseof acidic forms of compounds, be prepared from inorganic or organicbases. Frequently, the compounds are prepared or used aspharmaceutically acceptable salts prepared as addition products ofpharmaceutically acceptable acids or bases. Suitable pharmaceuticallyacceptable acids and bases are well-known in the art, such ashydrochloric, sulphuric, hydrobromic, acetic, lactic, citric, ortartaric acids for forming acid addition salts, and potassium hydroxide,sodium hydroxide, ammonium hydroxide, caffeine, various amines forforming basic salts. Methods for preparation of the appropriate saltsare well-established in the art.

Representative acid addition salts include acetate, adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,among others. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, and magnesium, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, andethylamine.

The term “polypeptide” as used herein refers to a string of at least twoamino acids attached to one another by a peptide bond. In someembodiments, a polypeptide may include at least 3-5 amino acids, each ofwhich is attached to others by way of at least one peptide bond. Thoseof ordinary skill in the art will appreciate that polypeptides caninclude one or more “non-natural” amino acids or other entities thatnonetheless are capable of integrating into a polypeptide chain. In someembodiments, a polypeptide may be glycosylated, e.g., a polypeptide maycontain one or more covalently linked sugar moieties. In someembodiments, a single “polypeptide” (e.g., an antibody polypeptide) maycomprise two or more individual polypeptide chains, which may in somecases be linked to one another, for example by one or more disulfidebonds or other means.

The term “radioconjugate,” as used herein, refers to any conjugate thatincludes a radioisotope or radionuclide, such as any of theradioisotopes or radionuclides described herein.

The term “radioimmunoconjugate,” as used herein, refers to anyimmunoconjugate that includes a radioisotope or radionuclide, such asany of the radioisotopes or radionuclides described herein.

The term “radioimmunotherapy,” as used herein, refers a method of usinga radioimmunoconjugate to produce a therapeutic effect. In someembodiments, radioimmunotherapy may include administration of aradioimmunoconjugate to a subject in need thereof, whereinadministration of the radioimmunoconjugate produces a therapeutic effectin the subject. In some embodiments, radioimmunotherapy may includeadministration of a radioimmunoconjugate to a cell, whereinadministration of the radioimmunoconjugate kills the cell. Whereinradioimmunotherapy involves the selective killing of a cell, in someembodiments the cell is a cancer cell in a subject having cancer.

As used herein, the term “radionuclide,” refers to an atom capable ofundergoing radioactive decay (e.g., ³H, 14C ¹⁵N, ¹⁸F, ³⁵S, ⁴⁷Sc, 55Co,⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁸⁹Zr, ⁸⁶Y ⁸⁷Y, ⁹⁰Y ⁹⁷Ru,⁹⁹Tc, ^(99m)Tc ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁴⁹Pm,¹⁴⁹Tb, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁸Au, ¹⁹⁹Au, ²⁰³Pb, ²¹¹At,²¹²Pb, ²¹²Bi, ²¹³Bi, ²²³Ra, ²²⁵Ac, ²²⁷Th, ²²⁹Th, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁸²Rb,^(117m)Sn ²⁰¹Tl). The terms radioactive nuclide, radioisotope, orradioactive isotope may also be used to describe a radionuclide.Radionuclides may be used as detection agents, as described above. Insome embodiments, the radionuclide is an alpha-emitting radionuclide.

By “subject” is meant a human or non-human animal (e.g., a mammal).

By “substantial identity” or “substantially identical” is meant apolypeptide sequence that has the same polypeptide sequence,respectively, as a reference sequence, or has a specified percentage ofamino acid residues, respectively, that are the same at thecorresponding location within a reference sequence when the twosequences are optimally aligned. For example, an amino acid sequencethat is “substantially identical” to a reference sequence has at least50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identity to the reference amino acid sequence. For polypeptides, thelength of comparison sequences will generally be at least 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90, 100, 150,200, 250, 300, or 350 contiguous amino acids (e.g., a full-lengthsequence). Sequence identity may be measured using sequence analysissoftware, e.g., on the default setting (e.g., Sequence Analysis SoftwarePackage of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Suchsoftware may match similar sequences by assigning degrees of homology tovarious substitutions, deletions, and other modifications.

The term “targeting moiety” as used herein refers to any molecule or anypart of a molecule that binds to a given target. In some embodiments,the targeting moiety is a protein or polypeptide such as an antibody orantigen binding fragment thereof, a nanobody, an affibody, or aconsensus sequence from a Fibronectin type III domain.

The term “therapeutic moiety” as used herein refers to any molecule orany part of a molecule that confers a therapeutic benefit. In someembodiments, the therapeutic moiety is a protein or polypeptide, e.g.,an antibody, an antigen-binding fragment thereof. In some embodiments,the therapeutic moiety is a small molecule.

As used herein, and as well understood in the art, “to treat” acondition or “treatment” of the condition (e.g., the conditionsdescribed herein such as cancer) is an approach for obtaining beneficialor desired results, such as clinical results. Beneficial or desiredresults can include, but are not limited to, alleviation or ameliorationof one or more symptoms or conditions; diminishment of extent ofdisease, disorder, or condition; stabilized (i.e., not worsening) stateof disease, disorder, or condition; preventing spread of disease,disorder, or condition; delay or slowing the progress of the disease,disorder, or condition; amelioration or palliation of the disease,disorder, or condition; and remission (whether partial or total),whether detectable or undetectable. In the context of cancer treatment,“ameliorating” may include, for example, reducing incidence ofmetastases, reducing tumor volume, reducing tumor vascularization and/orreducing the rate of tumor growth. “Palliating” a disease, disorder, orcondition means that the extent and/or undesirable clinicalmanifestations of the disease, disorder, or condition are lessenedand/or time course of the progression is slowed or lengthened, ascompared to the extent or time course in the absence of treatment.

As used herein, the term “tumor-associated antigen” means an antigenthat is present on tumor cells at a significantly greater amount than onnormal cells.

As used herein, the term “tumor-specific antigen” refers to an antigenthat is endogenously present only on tumor cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates relative tumor volume in the CT26 syngeneic mousetumor model after treatment with various checkpoint inhibitors.

FIG. 2 illustrates [¹⁷⁷Lu]-FPI-1755 biodistribution in the CT-26syngeneic mouse tumor model.

FIG. 3 illustrates the enhanced efficacy of [²²⁵Ac]-FPI-1792 inimmunocompetent mice vs. immunodeficient mice.

FIG. 4 illustrates synergy between [²²⁵Ac]-FPI-1792 and α-CTLA-4/PD-1treatment in the CT26 syngeneic mouse model.

FIG. 5 illustrates the development of protective immunity in[²²⁵Ac]-FPI-1792 (“[²²⁵Ac]-FPI-TAT” in the graph labels) treated miceupon CT26 re-challenge.

FIG. 6 illustrates cytokine response and T-cell recruitment after[²²⁵Ac]-FPI-1792 treatment.

FIG. 7 illustrates “humanized” IGF-1R model development.

It is to be understood that the figures are not necessarily drawn toscale, nor are the objects in the figures necessarily drawn to scale inrelationship to one another. The figures are depictions that areintended to bring clarity and understanding to various embodiments ofapparatuses, systems, and methods disclosed herein. Wherever possible,the same reference numbers will be used throughout the drawings to referto the same or like parts. Moreover, it should be appreciated that thedrawings are not intended to limit the scope of the present teachings inany way.

DETAILED DESCRIPTION

The present disclosure relates to combination therapies for inducing orimproving an immune response to cancer using radioimmunoconjugates andcheckpoint inhibitors. In some embodiments, use of methods disclosedherein results in treatment or amelioration of cancer.

In some embodiments, a lower effective dose of the radioimmunoconjugateand/or of the checkpoint inhibitor is used.

Radiolabelled targeting moieties (also known as radioimmunoconjugates)are designed to target a protein or receptor that is upregulated in adisease state and/or specific to diseased cells (e.g., tumor cells) todeliver a radioactive payload to damage and kill cells of interest.“Radioimmunotherapy” refers to this therapy when the targeting moietycomprises an antibody, typically a monoclonal antibody. Radioactivedecay of the payload produces an alpha, beta, or gamma particle or Augerelectron that can cause direct effects to DNA (such as single or doublestranded DNA breaks) or indirect effects such as by-stander or crossfireeffects.

Radioimmunoconjugates typically contain a biological targeting moiety(e.g., an antibody or antigen binding fragment thereof that specificallybinds to a molecule expressed on or by a tumor, e.g., IGF-1R orTEM-1/endosialin), a chelating moiety or a metal complex of a chelatingmoiety (e.g., comprising a radioisotope), and a linker. Conjugates maybe formed by appending a bifunctional chelate to the biologicaltargeting molecule so that structural alterations are minimal whilemaintaining target affinity. A radioimmunoconjugate may be formed byradiolabelling such a conjugate.

Bifunctional chelates structurally contain a chelate, a linker, and across-linking group. When developing new bifunctional chelates, mostefforts focus around the chelating portion of the molecule. Severalexamples of bifunctional chelates have been described with variouscyclic and acyclic structures conjugated to a targeted moiety.[Bioconjugate Chem. 2000, 11, 510-519, Bioconjugate Chem. 2012, 23,1029-1039, Mol Imaging Biol (2011) 13:215-221, Bioconjugate Chem. 2002,13, 110-115].

Radioimmunoconjugates

Radioimmunoconjugates suitable for use in accordance with the presentdisclosure generally have the structure of Formula I-a:

A-L-B  Formula I-a

wherein A is a chelating moiety or metal complex thereof),

wherein B is a targeting moiety, and

wherein L is a linker.

In some embodiments, the radioimmunoconjugate comprises the followingstructure:

wherein B is the targeting moiety.

Targeting Moieties

Targeting moieties include any molecule or any part of a molecule thatis capable of binding to a given target. In some embodiments, thetargeting moiety comprises a protein or polypeptide. In someembodiments, the targeting moiety is selected from the group consistingof antibodies or antigen binding fragments thereof, nanobodies,affibodies, and consensus sequences from Fibronectin type III domains(e.g., Centyrins or Adnectins). In some embodiments, a moiety is both atargeting and a therapeutic moiety, i.e., the moiety is capable ofbinding to a given target and also confers a therapeutic benefit.

Antibodies Antibodies typically comprise two identical light polypeptidechains and two identical heavy polypeptide chains linked together bydisulfide bonds. The first domain located at the amino terminus of eachchain is variable in amino acid sequence, providing the antibody-bindingspecificities of each individual antibody. These are known as variableheavy (VH) and variable light (VL) regions. The other domains of eachchain are relatively invariant in amino acid sequence and are known asconstant heavy (CH) and constant light (CL) regions. Light chainstypically comprise one variable region (VL) and one constant region(CL). An IgG heavy chain includes a variable region (VH), a firstconstant region (CH1), a hinge region, a second constant region (CH2),and a third constant region (CH3). In IgE and IgM antibodies, the heavychain includes an additional constant region (CH4).

Antibodies described herein can include, for example, monoclonalantibodies, polyclonal antibodies, multispecific antibodies, humanantibodies, humanized antibodies, camelid antibodies, chimericantibodies, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), andanti-idiotypic (anti-Id) antibodies, and antigen-binding fragments ofany of the above. In some embodiments, the antibody or antigen-bindingfragment thereof is humanized. In some embodiments, the antibody orantigen-binding fragment thereof is chimeric. Antibodies can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2) or subclass.

The term “antigen binding fragment” of an antibody, as used herein,refers to one or more fragments of an antibody that retain the abilityto specifically bind to an antigen. Examples of binding fragmentsencompassed within the term “antigen binding fragment” of an antibodyinclude a Fab fragment, a F(ab′)₂ fragment, a Fd fragment, a Fvfragment, a scFv fragment, a dAb fragment (Ward et al., (1989) Nature341:544-546), and an isolated complementarity determining region (CDR).In some embodiments, an “antigen binding fragment” comprises a heavychain variable region and a light chain variable region. These antibodyfragments can be obtained using conventional techniques known to thosewith skill in the art, and the fragments can be screened for utility inthe same manner as are intact antibodies.

Antibodies or fragments described herein can be produced by any methodknown in the art for the synthesis of antibodies (see, e.g., Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Brinkman et al., 1995, J. Immunol. Methods182:41-50; WO 92/22324; WO 98/46645). Chimeric antibodies can beproduced using the methods described in, e.g., Morrison, 1985, Science229:1202, and humanized antibodies by methods described in, e.g., U.S.Pat. No. 6,180,370.

Additional antibodies described herein are bispecific antibodies andmultivalent antibodies, as described in, e.g., Segal et al., J. Immunol.Methods 248:1-6 (2001); and Tutt et al., J. Immunol. 147: 60 (1991).

Insulin-Like Growth Factor 1 (IGF-1R) Antibodies

Insulin-like growth factor 1 receptor is a transmembrane protein foundon the surface of human cells activated by insulin-like growth factor 1(IGF-1) and 2 (IGF-2). In some embodiments, radioimmunoconjugatescomprise antibodies against insulin-like growth factor-1 receptor(IGF-1R). Although not a typical oncogene, IGF-1R promotes initiationand progression of cancer, playing a critical role in mitogenictransformation and maintenance of the transformed phenotype. IGF-1R hasbeen associated with development of multiple common cancers includingbreast, lung (e.g., non-small lung), liver, prostate, pancreas, ovarian,colon, melanoma, adrenocortical carcinoma, and various types ofsarcomas. IGF-1R signaling stimulates tumour cell proliferation andmetabolism, supports angiogenesis, and confers protection fromapoptosis. It affects metastatic factors (e.g., HIF-1 dependent hypoxiasignaling), anchorage independent growth, as well as growth and survivalof tumour metastases after extravasation. IGF-1R has also beenimplicated in the development, maintenance and enrichment of therapeuticresistant cancer stem cell populations.

Despite the abundance of data implicating IGF-1R's role in cancer,therapeutics targeting IGF-1R have yet to demonstrate a significantimpact on disease. There has been much speculation for this lack ofefficacy including the inability to identify appropriate biomarkers forpatient identification, complexity and interdependency of the IGF-1/IRsignaling pathway and the development of other growth hormonecompensatory mechanisms [Beckwith and Yee, Mol Endocrinol, November2015, 29(11):1549-1557]. Radioimmunotherapy, however, may provide aviable mechanism for treating cancers overexpressing the IGF-1 receptorby utilizing the ability of IGF-1R to undergo antibody triggeredinternalization and lysosomal degradation to deliver targetedradioisotopes inside cancer cells. Internalization and lysosomaldegradation of an IGF-1R targeted radioimmunoconjugate prolongs theresidence time of the delivered radioisotope inside cancer cells,thereby maximizing the potential for a cell killing emission to occur.In the case of actinium-225, which yields 4 alpha particles per decaychain, cell death can be accomplished by as little as 1 atom ofradionuclide delivered per cell [Sgouros, et al. J Nucl Med. 2010,51:311-2]. Cell killing due to direct DNA impact and breakage by analpha particle may occur in the targeted cell or in a radius of 2 or 3non-targeted cells for a given alpha particle decay. In addition tohaving very high potential anti-tumour potency, IGF-1R targetedradioimmunoconjugates may not generate mechanistic resistance as they donot rely on blocking ligand binding to the receptor to inhibit theoncologic process, as needed with a therapeutic antibody.

Several IGF-1R antibodies have been developed and investigated for thetreatment of various types of cancers, including figitumumab,cixutumumab, ganitumab, AVE1642 (also known as humanized EM164 andhuEM164), BlIB002, robatumumab, and teprotumumab. After binding toIGF-1R, these antibodies are internalized into the cell and degraded bylysosomal enzymes. The combination of overexpression on tumor cells andinternalization offers the possibility of delivering detection agentsdirectly to the tumor site while limiting the exposure of normal tissuesto toxic agents.

The CDRs of the light chain variable region of AVE1642 have thesequences:

SEQ ID NO: 1 (CDR-L1) RSSQSIVHSNVNTYLE SEQ ID NO: 2 (CDR-L2) KVSNRFSSEQ ID NO: 3 (CDR-L3) FQGSHVPPT

The light chain variable region of AVE1642 has the sequence:

SEQ ID NO: 4 DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK

The CDRs of the heavy chain variable region of AVE1642 have thesequences:

SEQ ID NO: 5 (CDR-H1) SYWMH SEQ ID NO: 6 (CDR-H2) GEINPSNGRTNY NQKFQGSEQ ID NO: 7 (CDR-H3) GRPDYYGSSKWY FDV

The heavy chain variable region of AVE1642 has the sequence:

SEQ ID NO: 8 QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPSNGRTNYNQKFQGKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGRPDYYGSSKWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG

Endosialin (TEM-1) Antibodies

Endosialin, also known as TEM-1 or CD-248, is an antigen expressed bytumor-associated endothelial cells, stromal cells, and pericytes.

Examples of endosialin antibodies include hMP-E-8.3 (disclosed in WO2017/134234, the entire contents of which are incorporated by referenceherein) and ontuxizumab (MORAb-004).

In some embodiments, the endosialin antibody or antigen-binding fragmentthereof recognizes an epitope having an amino acid sequence ofSRDHQIPVIAAN (SEQ ID NO: 9).

In some embodiments, the heavy chain variable region of the endosialinantibody or antibody-binding fragment thereof comprises thecomplementarity determining regions (CDRs) having the followingsequences:

(SEQ ID NO: 10) CDR-H1: GYGVN or (SEQ ID NO: 11) GFSLTGYGVN(SEQ ID NO: 12) CDR-H2: MIWVDGSTDYNSALKS (SEQ ID NO: 13)CDR-H3: GGYGAMDY

In some embodiments, the light chain variable region of the endosialinantibody or antibody-binding fragment thereof comprises thecomplementarity determining regions (CDRs) having the followingsequences:

(SEQ ID NO: 14) CDR-L1: HASQNINVWLT (SEQ ID NO: 15) CDR-L2: KASNLHT(SEQ ID NO: 16) CDR-L3: QQGQSYPWT

In some embodiments, the endosialin antibody or antigen-binding fragmentthereof is a humanized antibody.

In some embodiments, the heavy chain variable region of the endosialinantibody or antigen-binding fragment thereof comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 17, 18, 19, or20:

Humanized VH1: (SEQ ID NO: 17)QVQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPGKGLEWIGMIWVDGSTDYNSALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGY GAMDYWGQGTLVTVSSHumanized VH2: (SEQ ID NO: 18)QVQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPEKGLEWIGMIWVDGSTDYNSALKSRVNISVDTSKNQFSLKLSSVTAADTAVYYCARGGY GAMDYWGQGTLVTVSSHumanized VH3: (SEQ ID NO: 19)QLQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPGKGLEWIGMIWVDGSTDYNSALKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARGGY GAMDYWGQGTLVTVSSHumanized VH4: (SEQ ID NO: 20)QLQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPEKGLEWIGMIWVDGSTDYNSALKSRVNISVDKSKNQFSLKLSSVTAADTAVYYCARGGY GAMDYWGQGTLVTVSS

In some embodiments, the light chain variable region of the endosialinantibody or antigen-binding fragment thereof comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 21, 22, 23, or24:

Humanized VL1: (SEQ ID NO: 21)DIQMTQSPSSVSASVGDRVTITCHASQNINVWLTWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPWTFGG GTKLEIKHumanized VL2: (SEQ ID NO: 22)DIQMTQSPSTLSASVGDRVTITCHASQNINVWLTWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQGQSYPWTFGG GTKLEIKHumanized VL3: (SEQ ID NO: 23)DIQMTQSPSSLSASVGDRVTITCHASQNINVWLTWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGQSYPWTFGG GTKLEIKHumanized VL4: (SEQ ID NO: 24)DIQMTQSPSSLSASVGDRVTITCHASQNINVWLTWYQQKPEKAPKSLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPWTFGG GTKLEIK

Nanobodies

Nanobodies are antibody fragments consisting of a single monomericvariable antibody domain. Nanobodies may also be referred to assingle-domain antibodies. Like antibodies, nanobodies bind selectivelyto a specific antigen. Nanobodies may be heavy-chain variable domains orlight chain domains. Nanobodies may occur naturally or be the product ofbiological engineering. Nanobodies may be biologically engineered bysite-directed mutagenesis or mutagenic screening (e.g., phage display,yeast display, bacterial display, mRNA display, ribosome display).

Affibodies

Affibodies are polypeptides or proteins engineered to bind to a specificantigen. As such, affibodies may be considered to mimic certainfunctions of antibodies. Affibodies may be engineered variants of theB-domain in the immunoglobulin-binding region of staphylococcal proteinA. Affibodies may be engineered variants of the Z-domain, a B-domainthat has lower affinity for the Fab region. Affibodies may bebiologically engineered by site-directed mutagenesis or mutagenicscreening (e.g., phage display, yeast display, bacterial display, mRNAdisplay, ribosome display).

Affibody molecules showing specific binding to a variety of differentproteins (e.g. insulin, fibrinogen, transferrin, tumor necrosisfactor-α, IL-8, gp120, CD28, human serum albumin, IgA, IgE, IgM, HER2and EGFR) have been generated, demonstrating affinities (K_(d)) in theμM to μM range.

Fibronectin Type III Domains

The Fibronectin type III domain is an evolutionarily conserved proteindomain found in a wide-variety of extracellular proteins. TheFibronectin type III domain has been used as a molecular scaffold toproduce molecules capable of selectively binding a specific antigen.Variants of the Fibronectin type III domains (FN3) that have beenengineered for selective-binding may also be referred to as monobodies.FN3 domains may be biologically engineered by site-directed mutagenesisor mutagenic screening (e.g., CIS-display, phage display, yeast display,bacterial display, mRNA display, ribosome display).

Modified Polypeptides

Polypeptides used in accordance with the disclosure may have a modifiedamino acid sequence. Modified polypeptides may be substantiallyidentical to the corresponding reference polypeptide (e.g., the aminoacid sequence of the modified polypeptide may have at least 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity tothe amino acid sequence of the reference polypeptide). In certainembodiments, the modification does not destroy significantly a desiredbiological activity (e.g., binding to IGF-1R or to endosialin). Themodification may reduce (e.g., by at least 5%, 10%, 20%, 25%, 35%, 50%,60%, 70%, 75%, 80%, 90%, or 95%), may have no effect, or may increase(e.g., by at least 5%, 10%, 25%, 50%, 100%, 200%, 500%, or 1000%) thebiological activity of the original polypeptide. The modifiedpolypeptide may have or may optimize a characteristic of a polypeptide,such as in vivo stability, bioavailability, toxicity, immunologicalactivity, immunological identity, and conjugation properties.

Modifications include those by natural processes, such aspost-translational processing, or by chemical modification techniquesknown in the art. Modifications may occur anywhere in a polypeptideincluding the polypeptide backbone, the amino acid side chains and theamino- or carboxy-terminus. The same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide,and a polypeptide may contain more than one type of modification.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched, and branchedcyclic polypeptides may result from post-translational natural processesor may be made synthetically. Other modifications include pegylation,acetylation, acylation, addition of acetomidomethyl (Acm) group,ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation,carboxyethylation, esterification, covalent attachment to flavin,covalent attachment to a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of drug,covalent attachment of a marker (e.g., fluorescent or radioactive),covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphatidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent crosslinks, formation ofcystine, formation of pyroglutamate, formylation, gamma-carboxylation,glycosylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation and ubiquitination.

A modified polypeptide can also include an amino acid insertion,deletion, or substitution, either conservative or non-conservative(e.g., D-amino acids, desamino acids) in the polypeptide sequence (e.g.,where such changes do not substantially alter the biological activity ofthe polypeptide). In particular, the addition of one or more cysteineresidues to the amino or carboxy-terminus of a polypeptide canfacilitate conjugation of these polypeptides by, e.g., disulfidebonding. For example, a polypeptide can be modified to include a singlecysteine residue at the amino-terminus or a single cysteine residue atthe carboxy-terminus. Amino acid substitutions can be conservative(i.e., wherein a residue is replaced by another of the same general typeor group) or non-conservative (i.e., wherein a residue is replaced by anamino acid of another type). In addition, a naturally occurring aminoacid can be substituted for a non-naturally occurring amino acid (i.e.,non-naturally occurring conservative amino acid substitution or anon-naturally occurring non-conservative amino acid substitution).

Polypeptides made synthetically can include substitutions of amino acidsnot naturally encoded by DNA (e.g., non-naturally occurring or unnaturalamino acid). Examples of non-naturally occurring amino acids includeD-amino acids, N-protected amino acids, an amino acid having anacetylaminomethyl group attached to a sulfur atom of a cysteine, apegylated amino acid, the omega amino acids of the formulaNH₂(CH₂)_(n)COOH wherein n is 2-6, neutral nonpolar amino acids, such assarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, andnorleucine. Phenylglycine may substitute for Trp, Tyr, or Phe;citrulline and methionine sulfoxide are neutral nonpolar, cysteic acidis acidic, and ornithine is basic. Proline may be substituted withhydroxyproline and retain the conformation conferring properties.

Analogs may be generated by substitutional mutagenesis and retain thebiological activity of the original polypeptide. Examples ofsubstitutions identified as “conservative substitutions” are shown inTable 1. If such substitutions result in a change not desired, thenother type of substitutions, denominated “exemplary substitutions” inTable 1, or as further described herein in reference to amino acidclasses, are introduced and the products screened.

TABLE 1 Amino acid substitutions Original Conservative residue Exemplarysubstitution substitution Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln,Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser SerGln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Asn, Gln, Lys,Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, norleucine Leu Leu (L)Norleucine, Ile, Val, Met, Ala, Phe Ile Lys (K) Arg, Gln, Asn Arg Met(M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala Leu Pro (P) Gly Gly Ser(S) Thr Thr Thr (T) Ser Ser Trp (VV) Tyr Tyr Tyr (Y) Trp, Phe, Thr, SerPhe Val (V) Ile, Leu, Met, Phe, Ala, norleucine Leu

Substantial modifications in function or immunological identity areaccomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain.

Chelating Moieties and Metal Complexes Thereof

Chelating Moieties

Examples of suitable chelating moieties include, but are not limited to,DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA(1R,4R,7R,10R)-α, α′, α″,α′″-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, DOTAM(1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane),DOTPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra propionic acid),DO3AM-acetic acid(2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)aceticacid), DOTA-GA anhydride(2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triaceticacid, DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid)), DOTMP(1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylene phosphonic acid,DOTA-4AMP(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonicacid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diaceticacid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP(1,4,7-triazacyclononane-1,4,7-tri(methylene phosphonic acid), TETPA(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetra acetic acid), HEHA(1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid),PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N′,N″,N′″,N ″″-pentaaceticacid), H₄octapa(N,N′-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N′-diaceticacid), H₂dedpa (1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane),H₆phospa(N,N′-(methylenephosphonate)-N,N′-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane),TTHA (triethylenetetramine-N,N,N′,N″,N′″, N′″-hexaacetic acid), DO2P(tetraazacyclododecane dimethanephosphonic acid), HP-DO3A(hydroxypropyltetraazacyclododecanetriacetic acid), EDTA(ethylenediaminetetraacetic acid), Deferoxamine, DTPA(diethylenetriaminepentaacetic acid), DTPA-BMA(diethylenetriaminepentaacetic acid-bismethylamide), HOPO (octadentatehydroxypyridinones), or porphyrins.

In some embodiments, radioimmunoconjugates comprise a metal complex of achelating moiety. For example, chelating groups may be used in metalchelate combinations with metals, such as manganese, iron, andgadolinium and isotopes (e.g., isotopes in the general energy range of60 to 4,000 keV), such as any of the radioisotopes and radionuclidesdiscussed herein.^(2, 2 2), In some embodiments, chelating moieties areuseful as detection agents, and radioimmunoconjugates comprising suchdetectable chelating moieties can therefore be used as diagnostic ortheranostic agents.

Radioisotopes and Radionuclides

In some embodiments, the metal complex comprises a radionuclide.Examples of suitable radioisotopes and radionuclides include, but arenot limited to, ³H, ¹⁴C, ¹⁵N, ¹⁸F, ³⁵S, ⁴⁷Sc, ⁵⁵Co, ⁶⁰Cu, ⁶¹Cu, ⁶²Cu,⁶⁴Cu, ⁶⁶Ga, ⁶⁷Ga, ⁶⁷Cu, ⁶⁸Ga, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁸²Rb, ⁸⁹Zr, ⁸⁶Y, ⁸⁷Y,⁹⁰Y, ⁹⁷Ru, ⁹⁹Tc, ^(99m)Tc, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I,¹⁴⁹Pm, ¹⁴⁹Tb, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ^(117m)Sn, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁸Au,¹⁹⁹Au, ²⁰¹TI, ²⁰³Pb, ²¹¹At, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²³Ra, ²²⁵Ac, ²²⁷Th,and ²²⁹Th.

In some embodiments, the radionuclide is an alpha emitter, e.g.,Astatine-211 (²¹¹At), Bismuth-212 (²¹²Bi), Bismuth-213 (²¹³Bi),Actinium-225 (²²⁵Ac), Radium-223 (²²³Ra), Lead-212 (²¹²Pb), Thorium-227(²²⁷Th), or Terbium-149 (¹⁴⁹Tb).

Linkers

In some embodiments, the linker is as shown within the structure ofFormula I-b, as that part of Formula I-b absent A and B:

A-L¹-(L²)_(n)-B   Formula I-b

(A and B are as defined in Formula I-a.)

Thus, in some embodiments, the linker is -L¹-(L²)_(n)-,

-   -   wherein L¹ is optionally substituted C₁-C₆ alkyl, substituted        C₁-C₆ heteroalkyl, substituted aryl or heteroaryl;    -   n is 1-5; and    -   each L², independently, has the structure:

(—X¹-L³-Z¹—)   Formula II

wherein is X¹ is C═O(NR¹), C═S(NR¹), OC═O(NR¹), NR¹C═O(O), NR¹C═O(NR¹),—CH₂PhC═O(NR¹), —CH₂Ph(NH)C═S(NR¹), O, or NR¹; and each R¹ independentlyis H or optionally substituted C₁-C₆ alkyl or optionally substitutedC₁-C₆ heteroalkyl, substituted aryl or heteroaryl, in which C₁-C₆ alkylcan be substituted by oxo (═O), heteroaryl, or a combination thereof;L³ is optionally substituted C₁-C₅₀ alkyl or optionally substitutedC₁-C₅₀ heteroalkyl or C₅-C₂₀ polyethylene glycol; Z¹ is CH₂, C═O, C═S,OC═O, NR¹C═O, NR¹ and R¹ is a hydrogen or optionally substituted C₁-C₆alkyl, pyrrolidine-2,5-dione.

Cross-Linking Groups

In some embodiments, radioimmunoconjugates comprise a cross-linkinggroup instead of or in addition to the targeting moiety or therapeuticmoiety (e.g., B in Formula I comprises a cross-linking group).

A cross-linking group is a reactive group that is able to join two ormore molecules by a covalent bond. Cross-linking groups may be used toattach the linker and chelating moiety to a therapeutic or targetingmoiety. Cross-linking groups may also be used to attach the linker andchelating moiety to a target in vivo. In some embodiments, thecross-linking group is an amino-reactive, methionine reactive orthiol-reactive cross-linking group, or a sortase-mediated coupling. Insome embodiments, the amino-reactive or thiol-reactive cross-linkinggroup comprises an activated ester such as a hydroxysuccinimide ester,2,3,5,6-tetrafluorophenol ester, 4-nitrophenol ester or an imidate,anhydride, thiol, disulfide, maleimide, azide, alkyne, strained alkyne,strained alkene, halogen, sulfonate, haloacetyl, amine, hydrazide,diazirine, phosphine, tetrazine, isothiocyanate, or oxaziridine. In someembodiments, the sortase recognition sequence may comprise of a terminalglycine-glycine-glycine (GGG) and/or LPTXG amino acid sequence, where Xis any amino acid. A person having ordinary skill in the art willunderstand that the use of cross-linking groups is not limited to thespecific constructs disclosed herein, but rather may include other knowncross-linking groups.

Checkpoint Inhibitors

In some embodiments, a checkpoint inhibitor is co-administered with aradioimmunoconjugate. Generally, suitable checkpoint inhibitors inhibitan immune suppressive checkpoint protein. In some embodiments, thecheckpoint inhibitor inhibits a protein selected from the groupconsisting of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4),programmed death 1 (PD-1), programmed death ligand-1 (PD-L1), LAG-3, Tcell immunoglobulin mucin 3 (TIM-3), and killer immunoglobulin-likereceptors (KIRs).

For example, in some embodiments, the checkpoint inhibitor is capable ofbinding to CTLA-4, PD-1, or PD-L1. In some embodiments, the checkpointinhibitor interferes with the interaction (e.g., interferes withbinding) between PD-1 and PD-L1.

In some embodiments, the checkpoint inhibitor is a small molecule.

In some embodiments, the checkpoint inhibitor is an antibody orantigen-binding fragment thereof, e.g., a monclonal antibody. In someembodiments, the checkpoint inhibitor is a human or humanized antibodyor antigen-binding fragment thereof. In some embodiments, the checkpointinhibitor is a mouse antibody or antigen-binding fragment thereof.

In some embodiments, the checkpoint inhibitor is a CTLA-4 antibody.Non-limiting examples of CTLA-4 antibodies include BMS-986218,BMS-986249, ipilimumab, tremelimumab (formerly ticilimumab, CP-675,206),MK-1308, and REGN-4659. An additional example of a CTLA-4 antibody is4F10-11, a mouse monoclonal antibody.

In some embodiments, the checkpoint inhibitor is a PD-1 antibody.Non-limiting examples of PD-1 antibodies include camrelizumab,cemiplumab, nivolumab, pembrolizumab, sintilimab, tislelizumab andtoripalimab. An additional example of a PD-1 antibody is RMP1-14, amouse monoclonal antibody.

In some embodiments, the checkpoint inhibitor is a PD-L1 antibody.Non-limiting examples of PD-L1 antibodies include atezolizumab,avelumab, and durvalumab.

In some embodiments, a combination of more than one checkpoint inhibitoris used. For example, in some embodiments, both a CTLA-4 inhibitor and aPD-1 or PD-L1 inhibitor is used.

Subjects

In some disclosed methods, a therapy (e.g., comprising a therapeuticagent) is administered to a subject. In some embodiments, the subject isa mammal, e.g., a human.

In some embodiments, the subject has received or is receiving anothertherapy. For example, in some embodiments, the subject has received oris receiving a radioimmunoconjugate. In some embodiments, the subjecthas received or is receiving a checkpoint inhibitor.

In some embodiments, the subject has cancer or is at risk of developingcancer. For example, the subject may have been diagnosed with cancer.The cancer may be a primary cancer or a metastatic cancer. Subjects mayhave any stage of cancer, e.g., stage I, stage II, stage III, or stageIV with or without lymph node involvement and with or withoutmetastases. Provided compositions may prevent or reduce further growthof the cancer and/or otherwise ameliorate the cancer (e.g., prevent orreduce metastases). In some embodiments, the subject does not havecancer but has been determined to be at risk of developing cancer, e.g.,because of the presence of one or more risk factors such asenvironmental exposure, presence of one or more genetic mutations orvariants, family history, etc. In some embodiments, the subject has notbeen diagnosed with cancer.

In some embodiments, the cancer is a solid tumor.

In some embodiments, the solid tumor cancer is breast cancer, non-smallcell lung cancer, small cell lung cancer, pancreatic cancer, head andneck cancer, prostate cancer, colorectal cancer, sarcoma, adrenocorticalcarcinoma, neuroendocrine cancer, Ewing's Sarcoma, multiple myeloma, oracute myeloid leukemia.

In some embodiments, the cancer is a non-solid (e.g., liquid (e.g.,hematologic)) cancer.

Administration and Dosage

Effective and Lower Effective Doses

The present disclosure provides combination therapies in which theamounts of each therapeutic may or may not be, on their own,therapeutically effective. For example, provided are methods comprisingadministering a first therapy and a second therapy in amounts thattogether are effective to treat or ameliorate a disorder, e.g., cancer.In some embodiments, at least one of the first and second therapy isadministered to the subject in a lower effective dose. In someembodiments, both the first and the second therapies are administered inlower effective doses.

In some embodiments, the first therapy comprises a radioimmunoconjugateand the second therapy comprises a checkpoint inhibitor.

In some embodiments, the first therapy comprises a checkpoint inhibitorand the second therapy comprises a radioimmunoconjugate.

In some embodiments, therapeutic combinations as disclosed herein areadministered to a subject in a manner (e.g., dosing amount and timing)sufficient to cure or at least partially arrest the symptoms of thedisorder and its complications. In the context of a single therapy (a“monotherapy”), an amount adequate to accomplish this purpose is definedas a “therapeutically effective amount,” an amount of a compoundsufficient to substantially improve at least one symptom associated withthe disease or a medical condition. The “therapeutically effectiveamount” typically varies depending on the therapeutic. For knowntherapeutic agents, the relevant therapeutically effective amounts maybe known to or readily determined by those of skill in the art.

For example, in the treatment of cancer, an agent or compound thatdecreases, prevents, delays, suppresses, or arrests any symptom of thedisease or condition would be therapeutically effective. Atherapeutically effective amount of an agent or compound is not requiredto cure a disease or condition but will provide a treatment for adisease or condition such that the onset of the disease or condition isdelayed, hindered, or prevented, or the disease or condition symptomsare ameliorated, or the term of the disease or condition is changed or,for example, is less severe or recovery is accelerated in an individual.For example, a treatment may be therapeutically effective if it causes acancer to regress or to slow the cancer's growth.

The dosage regimen (e.g., amounts of each therapeutic, relative timingof therapies, etc.) that is effective for these uses may depend on theseverity of the disease or condition and the weight and general state ofthe subject. For example, the therapeutically effective amount of aparticular composition comprising a therapeutic agent applied to mammals(e.g., humans) can be determined by the ordinarily-skilled artisan withconsideration of individual differences in age, weight, and thecondition of the mammal. Because certain conjugates of the presentdisclosure exhibit an enhanced ability to target cancer cells andresidualize, the dosage of these compounds can be lower than (e.g., lessthan or equal to about 90%, 75%, 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of) the equivalent dose ofrequired for a therapeutic effect of the unconjugated agent.Therapeutically effective and/or optimal amounts can also be determinedempirically by those of skill in the art. Thus, lower effective dosescan also be determined by those of skill in the art.

Single or multiple administrations of a composition (e.g., apharmaceutical composition comprising a therapeutic agent) can becarried out with dose levels and pattern being selected by the treatingphysician. The dose and administration schedule can be determined andadjusted based on the severity of the disease or condition in thesubject, which may be monitored throughout the course of treatmentaccording to the methods commonly practiced by clinicians or thosedescribed herein.

In the disclosed combination therapy methods, the first and secondtherapies may be administered sequentially or concurrently to a subject.For example, a first composition comprising a first therapeutic agentand a second composition comprising a second therapeutic agent may beadministered sequentially or concurrently to a subject. Alternatively oradditionally, a composition comprising a combination of a firsttherapeutic agent and a second therapeutic agent may be administered tothe subject.

In some embodiments, the radioimmunoconjugate is administered in asingle dose. In some embodiments, the radioimmunoconjugate isadministered more than once. When the radioimmunoconjugate isadministered more than once, the dose of each administration may be thesame or different.

In some embodiments, the checkpoint inhibitor is administered in asingle dose. In some embodiments, the checkpoint inhibitor isadministered more than once, e.g., at least twice, at least three times,etc. In some embodiments, the checkpoint inhibitor is administeredmultiple times according to a regular or semi-regular schedule, e.g.,once every approximately two weeks, once a week, twice a week, threetimes a week, or more than three times a week. When the checkpointinhibitor is administered more than once, the dose of eachadministration may be the same or different. For example, the checkpointinhibitor may be administered in an initial dose amount, and thensubsequent dosages of the checkpoint inhibitor may be higher or lowerthan the initial dose amount.

In some embodiments, the first dose of the checkpoint inhibitor isadministered at the same time as the first dose of theradioimmunoconjugate. In some embodiments, the first dose of thecheckpoint inhibitor is administered before the first dose ofradioimmunoconjugate. In some embodiments, the first dose of thecheckpoint inhibitor is administered after the first dose ofradioimmunoconjugate. In some embodiments, subsequent doses of thecheckpoint inhibitor are administered.

In some embodiments, radioimmunoconjugates (or a composition thereof)and checkpoint inhibitors (or a composition thereof) are administeredwithin 28 days (e.g., within 14, 7, 6, 5, 4, 3, 2, or 1 day(s)) of eachother.

In some embodiments, radioimmunoconjugates (or a composition thereof)and checkpoint inhibitors (or a composition thereof) are administeredwithin 90 days (e.g., within 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2,or 1 day(s)) of each other. In various embodiments the checkpointinhibitor is administered at the same time as radioimmunoconjugate. Invarious embodiments, the checkpoint inhibitor is administered multipletimes after the first administration of radioimmunoconjugate.

In some embodiments, compositions (such as compositions comprisingradioimmunoconjugates) are administered for radiation treatment planningor diagnostic purposes. When administered for radiation treatmentplanning or diagnostic purposes, compositions may be administered to asubject in a diagnostically effective dose and/or an amount effective todetermine the therapeutically effective dose.

In some embodiments, a first dose of disclosed conjugate or acomposition (e.g., pharmaceutical composition) thereof is administeredin an amount effective for radiation treatment planning, followedadministration of a combination therapy including a conjugate asdisclosed herein and another therapeutic.

Pharmaceutical compositions comprising one or more agents (e.g.,radioimmunoconjugates and/or checkpoint inhibitors) can be formulatedfor use in accordance with disclosed methods and systems in a variety ofdrug delivery systems. One or more physiologically acceptable excipientsor carriers can also be included in the composition for properformulation. Examples of suitable formulations are found in Remington'sPharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa.,17th ed., 1985. For a brief review of methods for drug delivery, see,e.g., Langer (Science 249:1527-1533, 1990).

Formulations

Pharmaceutical compositions may be formulated for parenteral,intranasal, topical, oral, or local administration, such as by atransdermal means, for prophylactic and/or therapeutic treatment.Pharmaceutical compositions can be administered parenterally (e.g., byintravenous, intramuscular, or subcutaneous injection), or by oralingestion, or by topical application or intraarticular injection atareas affected by the vascular or cancer condition. Examples ofadditional routes of administration include intravascular,intra-arterial, intratumor, intraperitoneal, intraventricular,intraepidural, as well as nasal, ophthalmic, intrascleral, intraorbital,rectal, topical, or aerosol inhalation administration. Also specificallycontemplated are sustained release administration, by such means asdepot injections or erodible implants or components. Suitablecompositions include compositions comprising include agents (e.g.,compounds as disclosed herein) dissolved or suspended in an acceptablecarrier, preferably an aqueous carrier, e.g., water, buffered water,saline, or PBS, among others, e.g., for parenteral administration.Compositions may contain pharmaceutically acceptable auxiliarysubstances to approximate physiological conditions, such as pH adjustingand buffering agents, tonicity adjusting agents, wetting agents, ordetergents, among others. In some embodiments, compositions areformulated for oral delivery; for example, compositions may containinert ingredients such as binders or fillers for the formulation of aunit dosage form, such as a tablet or a capsule. In some embodiments,compositions are formulated for local administration; for example,compositions may contain inert ingredients such as solvents oremulsifiers for the formulation of a cream, an ointment, a gel, a paste,or an eye drop.

Compositions may be sterilized, e.g., by conventional sterilizationtechniques, or sterile filtered. Aqueous solutions may be packaged foruse as is, or lyophilized, the lyophilized preparation being combinedwith a sterile aqueous carrier prior to administration. The pH of thepreparations typically will be between 3 and 11, more preferably between5 and 9 or between 6 and 8, and most preferably between 6 and 7, such as6 to 6.5. In some embodiments, compositions in solid form are packagedin multiple single dose units, each containing a fixed amount of theabove-mentioned agent or agents, such as in a sealed package of tabletsor capsules. In some embodiments, compositions in solid form arepackaged in a container for a flexible quantity, such as in a squeezabletube designed for a topically applicable cream or ointment.

Effects

In some embodiments, methods of the present disclosure result in atherapeutic effect. In some embodiments, the therapeutic effectcomprises an immune response, for example, an immune response comprisesan increase in T cells, e.g. CD8+ (e.g., IFNγ-producing CD8+ cells)and/or CD4+ cells. In some embodiments, the T cells comprise T cellsspecific for a tumor-associated antigen or tumor-specific antigenexpressed on the cancer being treated or ameliorated. In someembodiments, the increase in T cells is observed in the tumor relativeto the spleen.

In some embodiments, the step of administering results in at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, or atleast 70% of the total T cell population in a sample in the mammal beingspecific for the tumor-associated antigen or tumor-specific antigen. Insome embodiments, the sample is a tumor sample.

In some embodiments, the therapeutic effect comprises a decrease intumor volume, a stable tumor volume, or a reduced rate of increase intumor volume. In some embodiments, the therapeutic effect comprises adecreased incidence of recurrence or metastasis.

Other Agents

In some embodiments, disclosed methods further include administering anantiproliferative agent, radiation sensitizer, or an immunoregulatory orimmunomodulatory agent.

By “antiproliferative” or “antiproliferative agent,” as usedinterchangeably herein, is meant any anticancer agent, including thoseantiproliferative agents listed in Table 2, any of which can be used incombination with a radioimmunoconjugate to treat a condition ordisorder. Antiproliferative agents also include organo-platinumderivatives, naphtoquinone and benzoquinone derivatives, chrysophanicacid and anthroquinone derivatives thereof.

By “immunoregulatory agent” or “immunomodulatory agent,” as usedinterchangeably herein, is meant any immuno-modulator, including thoselisted in Table 2, any of which can be used in combination with aradioimmunoconjugate.

As used herein, “radiation sensitizer” includes any agent that increasesthe sensitivity of cancer cells to radiation therapy. Radiationsensitizers may include, but are not limited to, 5-fluorouracil, analogsof platinum (e.g., cisplatin, carboplatin, oxaliplatin), gemcitabine,EGFR antagonists (e.g., cetuximab, gefitinib), farnesyltransferaseinhibitors, COX-2 inhibitors, bFGF antagonists, and VEGF antagonists.

TABLE 2 Alkylating agents Busulfan Chlorambucil dacarbazine procarbazineifosfamide altretamine hexamethylmelamine estramustine phosphatethiotepa mechlorethamine dacarbazine streptozocin lomustine temozolomidecyclophosphamide Semustine Platinum agents spiroplatin lobaplatin(Aeterna) tetraplatin satraplatin (Johnson Matthey) ormaplatin BBR-3464(Hoffmann-La Roche) iproplatin Miriplatin picoplatin AP-5280 (Access)oxaliplatin cisplatin carboplatin Antimetabolites azacytidinetrimetrexate Floxuridine deoxycoformycin 2-chlorodeoxyadenosinepentostatin 6-mercaptopurine hydroxyurea 6-thioguanine decitabine(SuperGen) cytarabine clofarabine (Bioenvision) 2-fluorodeoxy cytidineirofulven (MGI Pharma) methotrexate DMDC (Hoffmann-La Roche) tomudexethynylcytidine (Taiho) fludarabine gemcitabine raltitrexed capecitabineTopoisomerase amsacrine exatecan mesylate (Daiichi) inhibitorsepirubicin quinamed (ChemGenex) etoposide gimatecan (Sigma-Tau)teniposide or mitoxantrone diflomotecan (Beaufour-Ipsen)7-ethyl-10-hydroxy-camptothecin TAS-103 (Taiho) dexrazoxanet(TopoTarget) elsamitrucin (Spectrum) pixantrone (Novuspharma) Edotecarinrebeccamycin analogue (Exelixis) Cositecan BBR-3576 (Novuspharma)Belotecan rubitecan (SuperGen) hydroxycamptothecin (SN-38) irinotecan(CPT-11) topotecan Antitumor antibiotics valrubicin azonafidetherarubicin anthrapyrazole idarubicin oxantrazole rubidazonelosoxantrone plicamycin Sabarubicin porfiromycin Epirubicin mitoxantrone(novantrone) mitoxantrone amonafide doxorubicin Antimitotic colchicineE7010 (Abbott) agents vinblastine PG-TXL (Cell Therapeutics) vindesineIDN 5109 (Bayer) dolastatin 10 (NCI) A 105972 (Abbott) rhizoxin(Fujisawa) A 204197 (Abbott) mivobulin (Warner-Lambert) LU 223651 (BASF)cemadotin (BASF) D 24851 (ASTAMedica) RPR 109881A (Aventis) ER-86526(Eisai) TXD 258 (Aventis) combretastatin A4 (BMS) epothilone B(Novartis) isohomohalichondrin-B (PharmaMar) T 900607 (Tularik) ZD 6126(AstraZeneca) T 138067 (Tularik) AZ10992 (Asahi) cryptophycin 52 (EliLilly) IDN-5109 (Indena) vinflunine (Fabre) AVLB (Prescient NeuroPharma)auristatin PE (Teikoku Hormone) azaepothilone B (BMS) BMS 247550 (BMS)BNP-7787 (BioNumerik) BMS 184476 (BMS) CA-4 prodrug (OXiGENE) BMS 188797(BMS) dolastatin-10 (NIH) taxoprexin (Protarga) CA-4 (OXiGENE) SB 408075(GlaxoSmithKline) docetaxel Vinorelbine vincristine Trichostatin Apaclitaxel Aromatase inhibitors aminoglutethimide YM-511 (Yamanouchi)atamestane (BioMedicines) formestane letrozole exemestane anastrazoleThymidylate pemetrexed (Eli Lilly) nolatrexed (Eximias) synthaseinhibitors ZD-9331 (BTG) CoFactor ™ (BioKeys) DNA antagoniststrabectedin (PharmaMar) edotreotide (Novartis) glufosfamide (BaxterInternational) mafosfamide (Baxter International) albumin + 32P (IsotopeSolutions) apaziquone (Spectrum thymectacin (NewBiotics)Pharmaceuticals) O6 benzyl guanine (Paligent) Farnesyltransferasearglabin (NuOncology Labs) tipifarnib (Johnson & Johnson) inhibitorslonafarnib (Schering-Plough) perillyl alcohol (DOR BioPharma)BAY-43-9006 (Bayer) Pump inhibitors CBT-1 (CBA Pharma) zosuquidartrihydrochloride (Eli Lilly) tariquidar (Xenova) biricodar dicitrate(Vertex) MS-209 (Schering AG) Histone tacedinaline (Pfizer)pivaloyloxymethyl butyrate (Titan) acetyltransferase SAHA (Aton Pharma)depsipeptide (Fujisawa) inhibitors MS-275 (Schering AG)Metalloproteinase Neovastat (Aeterna Laboratories) CMT-3 (CollaGenex)inhibitors marimastat (British Biotech) BMS-275291 (Celltech)Ribonucleoside gallium maltolate (Titan) tezacitabine (Aventis)reductase inhibitors triapine (Vion) didox (Molecules for Health) TNFalpha virulizin (Lorus Therapeutics) revimid (Celgene)agonists/antagonists CDC-394 (Celgene) Endothelin A atrasentan (Abbott)YM-598 (Yamanouchi) receptor antagonist ZD-4054 (AstraZeneca) Retinoicacid fenretinide (Johnson & Johnson) alitretinoin (Ligand) receptoragonists LGD-1550 (Ligand) Immuno-modulators interferon dexosome therapy(Anosys) oncophage (Antigenics) pentrix (Australian Cancer GMK(Progenics) Technology) adenocarcinoma vaccine (Biomira) ISF-154(Tragen) CTP-37 (AVI BioPharma) cancer vaccine (Intercell) IRX-2(Immuno-Rx) norelin (Biostar) PEP-005 (Peplin Biotech) BLP-25 (Biomira)synchrovax vaccines (CTL Immuno) MGV (Progenics) melanoma vaccine (CTLImmuno) β-alethine (Dovetail) p21 RAS vaccine (GemVax) CLL therapy(Vasogen) MAGE-A3 (GSK) Ipilimumab (BMS), nivolumab (BMS) CM-10 (cCamBiotherapeutics) abatacept (BMS) atezolizumab (Genentech) pembrolizumab(Merck) Hormonal and estrogens dexamethasone antihormonal agentsconjugated estrogens prednisone ethinyl estradiol methylprednisolonechlortrianisen prednisolone idenestrol aminoglutethimidehydroxyprogesterone caproate leuprolide medroxyprogesterone octreotidetestosterone mitotane testosterone propionate; P-04 (Novogen)fluoxymesterone 2-methoxyestradiol (EntreMed) methyltestosteronearzoxifene (Eli Lilly) diethylstilbestrol tamoxifen megestrol toremofinebicalutamide goserelin flutamide Leuporelin nilutamide bicalutamidePhotodynamic talaporfin (Light Sciences) Pd-bacteriopheophorbide (Yeda)agents Theralux (Theratechnologies) Motexafin lutetium motexafingadolinium hypericin (Pharmacyclics) Kinase Inhibitors imatinib(Novartis) EKB-569 (Wyeth) leflunomide (Sugen/Pharmacia) kahalide F(PharmaMar) ZD1839 (AstraZeneca) CEP-701 (Cephalon) erlotinib (OncogeneScience) CEP-751 (Cephalon) canertinib (Pfizer) MLN518 (Millenium)squalamine (Genaera) PKC412 (Novartis) SU5416 (Pharmacia) Phenoxodiol(Novogen) SU6668 (Pharmacia) C225 (ImClone) ZD4190 (AstraZeneca) rhu-Mab(Genentech) ZD6474 (AstraZeneca) MDX-H210 (Medarex) vatalanib (Novartis)2C4 (Genentech) PKI166 (Novartis) MDX-447 (Medarex) GW2016(GlaxoSmithKline) ABX-EGF (Abgenix) EKB-509 (Wyeth) IMC-1C11 (ImClone)trastuzumab (Genentech) Tyrphostins OSI-774 (Tarceva ™) Gefitinib(Iressa) CI-1033 (Pfizer) PTK787 (Novartis) SU11248 (Pharmacia) EMD72000 (Merck) RH3 (York Medical) Emodin Genistein Radicinol RadicinolVemurafenib (B-Raf enzyme Met-MAb (Roche) inhibitor, Daiichi Sankyo)SR-27897 (CCK A inhibitor, Sanofi-Synthelabo) ceflatonin (apoptosispromotor, ChemGenex) tocladesine (cyclic AMP agonist, Ribapharm)BCX-1777 (PNP inhibitor, BioCryst) alvocidib (CDK inhibitor, Aventis)ranpimase (ribonuclease stimulant, Alfacell) CV-247 (COX-2 inhibitor,Ivy Medical) galarubicin (RNA synthesis inhibitor, Dong-A) P54 (COX-2inhibitor, Phytopharm) tirapazamine (reducing agent, SRI CapCell ™(CYP450 stimulant, Bavarian Nordic) International) GCS-100 (gal3antagonist, GlycoGenesys) N-acetylcysteine (reducing agent, Zambon)G17DT immunogen (gastrin inhibitor, Aphton) R-flurbiprofen (NF-kappaBinhibitor, Encore) efaproxiral (oxygenator, Allos Therapeutics) 3CPA(NF-kappaB inhibitor, Active Biotech) PI-88 (heparanase inhibitor,Progen) seocalcitol (vitamin D receptor agonist, Leo) tesmilifene(histamine antagonist, YM 131-l-TM-601 (DNA antagonist, BioSciences)TransMolecular) histamine (histamine H2 receptor agonist, Maxim)eflornithine (ODC inhibitor, ILEX Oncology) tiazofurin (IMPDH inhibitor,Ribapharm) minodronic acid (osteoclast inhibitor, cilengitide (integrinantagonist, Merck KGaA) Yamanouchi) SR-31747 (IL-1 antagonist,Sanofi-Synthelabo) indisulam (p53 stimulant, Eisai) CCI-779 (mTOR kinaseinhibitor, Wyeth) aplidine (PPT inhibitor, PharmaMar) exisulind (PDE Vinhibitor, Cell Pathways) gemtuzumab (CD33 antibody, Wyeth Ayerst)CP-461 (PDE V inhibitor, Cell Pathways) PG2 (hematopoiesis enhancer,AG-2037 (GARFT inhibitor, Pfizer) Pharmagenesis) WX-UK1 (plasminogenactivator inhibitor, Wilex) Immunol ™ (triclosan oral rinse, Endo)PBI-1402 (PMN stimulant, ProMetic LifeSciences) triacetyluridine(uridine prodrug, Wellstat) bortezomib (proteasome inhibitor,Millennium) SN-4071 (sarcoma agent, Signature SRL-172 (T cell stimulant,SR Pharma) BioScience) TLK-286 (glutathione S transferase inhibitor,TransMID-107 ™ (immunotoxin, KS Biomedix) Telik) PCK-3145 (apoptosispromotor, Procyon) PT-100 (growth factor agonist, Point doranidazole(apoptosis promotor, Pola) Therapeutics) CHS-828 (cytotoxic agent, Leo)midostaurin (PKC inhibitor, Novartis) trans-retinoic acid(differentiator, NIH) bryostatin-1 (PKC stimulant, GPC Biotech) MX6(apoptosis promotor, MAXIA) CDA-II (apoptosis promotor, Everlife)apomine (apoptosis promotor, ILEX Oncology) SDX-101 (apoptosis promotor,Salmedix) urocidin (apoptosis promotor, Bioniche) rituximab (CD20antibody, Genentech Ro-31-7453 (apoptosis promotor, La Roche) carmustinebrostallicin (apoptosis promotor, Pharmacia) Mitoxantrone β-lapachoneBleomycin gelonin Absinthin cafestol Chrysophanic acid kahweol Cesiumoxides caffeic acid BRAF inhibitors, Tyrphostin AG PD-L1 inhibitors PD-1inhibitors MEK inhibitors CTLA-4 inhibitors bevacizumab sorafenibangiogenesis inhibitors dabrafenib

EXAMPLES Example 1. Single Agent Efficacy of Checkpoint Inhibitors inthe CT-26 Syngeneic Model was Observed

A single agent efficacy study of two checkpoint inhibitors (PD-1 andCTLA-4) was conducted in the CT-26 model, a murine colon carcinomamodel. It is known that these carcinomas are partially sensitive toα-PD-1 mAbs and sensitive to α-CTLA-4 mAbs. Mice were injected i.p. witheither 5 or 15 mg/kg i.p. of either the α-PD-1 mAb or the α-CTLA-4 mAb.The α-PD-1 mAb group was dosed twice a week for four weeks. The α-CTLA-4mAb group was dosed only 3 times a day, 3 days apart. CTLA-4 treatmentwas more efficacious than PD-1 treatment, as expected for this model. Inboth treatment groups, 5 mg/kg appeared to be the most efficacious dosefor impairing tumor growth. See FIG. 1. CD8+/CD4+ T cell recruitmentfollowing the different treatments is also measured usingimmunohistochemistry and flow cytometry techniques.

Example 2. [¹⁷⁷Lu]-FPI-1755 Biodistribution in the CT-26 Syngeneic Model

MAB391, a murine monoclonal antibody against IGF-1R, was conjugated withFPI-1397 (a bifunctional chelate) and radiolabeled with Lu-177 usingmethods well known in the art to form [¹⁷⁷Lu]-FPI-1755. The ability of[¹⁷⁷Lu]-FPI-1755 to target antigen expressing mouse IGF-1Roverexpressing tumors in vivo was demonstrated using the CT-26 syngeneicmodel. Tumor uptake was steady at 15-17% injected dose/g (ID/g) from24-96 hours post injection. See FIG. 2.

Example 3. Enhanced Efficacy of [²²⁵Ac]-FPI-1792 in Immunocompetent vs.in Immunodeficient Mice

MAB391, a murine monoclonal antibody against IGF-1R, was conjugated withFPI-1397 (a bifunctional chelate) and radiolabeled with [²²⁵Ac] usingstandard techniques to form [²²⁵Ac]-FPI-1792. An efficacy study of[²²⁵Ac]-FPI-1792 in immunocompetent and in immunodeficient mice wasconducted using a 400 nCi dose of [²²⁵Ac]-FPI-1792. It was found that[²²⁵Ac]-FPI-1792 had enhanced efficacy in reducing tumor volume in micewith an intact immune system relative to mice with no immune system. SeeFIG. 3.

Example 4. Synergy between [²²⁵Ac]-FPI-1792 and α-CTLA-4/PD-1 Treatmentin the CT26 Syngeneic Mouse Model

An in vivo synergy study was conducted to test the effect of[²²⁵Ac]-FPI-1792 (as described in Example 3) and checkpoint inhibitors,α-CTLA-4 and α-PD-1 antibodies, on relative tumor volume in the CT26mouse model. Mice treated either with the CTLA-4 inhibitor alone or thePD-1 inhibitor alone showed modest reductions in relative tumor volumewhen compared to the vehicle control groups. Mice treated with[²²⁵Ac]-FPI-1792 demonstrated greater reductions in tumor volumerelative to the vehicle control group or the groups administered CTLA-4inhibitor or PD-1 inhibitor alone. However, when [²²⁵Ac]-FPI-1792 wasco-administered with ether CTLA-4 or PD-1 or both, a synergistic effectwas seen-co-administration resulted in significantly smaller tumorvolume when compared to treatment with [²²⁵Ac]-FPI-1792, or whencompared to treatment with the CTLA-4 inhibitor or the PD-1 inhibitoralone. See FIG. 4.

Example 5. Development of Protective Immunity in [²²⁵Ac]-FPI-1792retreated Mice upon CT26 Re-Challenge

A re-challenge experiment was conducted to test the development ofprotective immunity in [²²⁵Ac]-FPI-1792 treated mice upon CT26re-challenge. Mice had been previously treated with either[²²⁵Ac]-FPI-1792 alone or in combination with an α-CTLA-4 or α-PD-1antibody. Naïve mice were used as controls. All mice previously treatedwith [²²⁵Ac]-FPI-1792+/−an anti-CTLA-4 or anti-PD-1 antibody wereprotected from tumor challenge, suggesting development of protective Tcell immunity. See FIG. 5.

Example 6. Cytokine Response and T-Cell Recruitment after[²²⁵Ac]-FPI-1792 Treatment

Cytokine response and T-cell recruitment after [²²⁵Ac]-FPI-1792treatment is measured. Mice were inoculated with 1×10⁶ CT26 cells. Micewere then treated with either [²²⁵Ac]-FPI-1792, the unconjugated MAB391antibody or vehicle. Samples from the tumor, spleen and blood plasma areanalyzed for the presence of cytokines at 24, 48, or 72 hours.Additional samples are taken from the tumor and spleen at 72 hours, 5days and 8 days for immunohistochemistry to assess the presence ofdifferent T-cell types. Finally, at 8 days, tumor-infiltratinglymphocytes are extracted, isolated and quantified using flow cytometry.See FIG. 6.

CT26 cells were stably transfected with human IGF-1R plasmid. Westernblot analysis was conducted for the presence of hIGF-1R for theselection of hIGF-1R expressing clones. See FIG. 7. The best clones arechosen based on both in vitro and in vivo characteristics. The resultingcell lines will be used as an immunocompetent mouse model for testing[²²⁵Ac]-FPI-1434 and other radioimmunoconjugates and additionalsynergies with immune checkpoint inhibitors.

Example 7. Combination Therapies Result in Increased Tumor-AssociatedAntigen-Specific CD8+ T Cells in Both the Spleen and Tumor Itself

Ac-TAB-199 is a radioimmunoconjugate comprising human monoclonal IGF-1Rantibody labeled with ²²⁵-Actinium. Combinations with Ac-TAB-199 andcheckpoint inhibitors (α-PD-1, α-CTLA-4, or both α-PD-1 and α-CTLA-4)were tested in the CT26 syngeneic mouse model. Mice were re-challengedwith CT26 cells at day 28 after initial tumor inoculation. CD8+ and CD4+T cell populations were assessed in both the spleen and the tumor afterre-challenge. In mice treated with Ac-TAB-199 and checkpoint inhibitors,both the spleen and the tumor exhibited the presence of CD8+ T-cells.Importantly, an increase in the CD8+ T-cell frequency in the tumor,relative to controls, was observed. These results suggest that thesecombination treatments lead to improved levels of therapeuticallyeffective CD8+ T cells.

Antigen-specific T-cells were detected and enumerated using an MHC classI tetramer assay. In this assay, MHC I molecules presenting an epitopespecific to CT26 cells are labelled with biotin. In the presence ofstreptavidin, these MHC I molecules tetramerize. CD8+ T cells specificfor the CD26 epitope are thereby labelled when their T-cell receptorsbind to MHC I/CT26 epitope complexes within tetramers. Based on tetrameranalysis, approximately 35%, 62%, and 75% of the CD8+ T cells wereantigen-specific in mice treated with Ac-TAB-199/α-CTLA-4,Ac-TAB-199/α-PD-1, and Ac-TAB-199/α-CTLA-4/α-PD-1, respectively.

EQUIVALENTS/OTHER EMBODIMENTS

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments described herein. Such equivalents are intended to beencompassed by the following claims.

1. A method of inducing an immune response to a tumor in a mammal, saidmethod comprising: (i) administering to the mammal aradioimmunoconjugate, wherein the mammal has received or is receivingone or more checkpoint inhibitors; (ii) administering to the mammal oneor more checkpoint inhibitors, wherein the mammal has received or isreceiving a radioimmunoconjugate; or (iii) administering the mammal oneor more checkpoint inhibitors at the same time as administering themammal a radioimmunoconjugate.
 2. The method of claim 1, said methodcomprising administering to a mammal one or more checkpoint inhibitors,wherein the mammal has received or is receiving a radioimmunoconjugate.3. The method of claim 1 or 2, wherein the one or more checkpointinhibitors is administered in a lower effective dose.
 4. The method ofany one of claims 1-3, wherein the radioimmunoconjugate is administeredin a lower effective dose.
 5. The method of claim 1, wherein theradioimmunoconjugate comprises (i) a targeting moiety, (ii) a linker,and (iii) a chelating moiety or a metal complex of a chelating moiety.6. The method of claim 5, wherein the targeting moiety is capable ofbinding to a tumor-associated antigen.
 7. The method of claim 6, whereinthe tumor-associated antigen is a tumor-specific antigen.
 8. The methodof claim 6 or 7, wherein the targeting moiety is an antibody or anantigen-binding fragment thereof.
 9. The method of claim 8, wherein theantibody or antigen-binding fragment thereof is an IGF-1R antibody or anantigen-binding fragment thereof.
 10. The method of claim 8, wherein theantibody or antigen-binding fragment thereof is an endosialin (TEM-1)antibody or an antigen-binding fragment thereof.
 11. The method of anyone of claims 5-10, wherein the radioimmunoconjugate comprises a metalcomplex of a chelating moiety.
 12. The method of claim 11, wherein themetal complex comprises a radionuclide.
 13. The method of claim 12,wherein the radionuclide is an alpha emitter.
 14. The method of claim13, wherein the radionuclide is an alpha emitter selected from the groupconsisting of Astatine-211 (²¹¹At), Bismuth-212 (²¹²Bi), Bismuth-213(²¹³Bi), Actinium-225 (²²⁵Ac), Radium-223 (²²³Ra), Lead-212 (²¹²Pb),Thorium-227 (²²⁷Th), and Terbium-149 (¹⁴⁹Tb).
 15. The method of claim14, wherein the radionuclide is ²²⁵Ac.
 16. The method of any one ofclaims 5-15, wherein the radioimmunoconjugate comprises the followingstructure:

wherein B is the targeting moiety.
 17. The method of any one of claims1-16, wherein the one or more checkpoint inhibitors comprise a PD-1inhibitor.
 18. The method of claim 17, wherein the PD-1 inhibitor is anantibody.
 19. The method of any one of claims 1-18, wherein the one ormore checkpoint inhibitors comprise an CTLA-4 inhibitor.
 20. The methodof claim 19, wherein the CTLA-4 inhibitor is antibody.
 21. The method ofany one of claims 1-16, wherein the one or more checkpoint inhibitorscomprises both a PD-1 inhibitor and a CTLA-4 inhibitor.
 22. The methodof any one of claims 1-21, wherein the mammal is a human.
 23. The methodof any one of claims 1-22, wherein the mammal is diagnosed with cancer.24. The method of claim 23, wherein the cancer is selected from thegroup comprising: breast cancer, non-small cell lung cancer, small celllung cancer, pancreatic cancer, head and neck cancer, prostate cancer,colorectal cancer, sarcoma, adrenocortical carcinoma, neuroendocrinecancer, Ewing's Sarcoma, multiple myeloma, or acute myeloid leukemia.25. The method of any one of claims 1-24, wherein mammal has at leastone solid tumor.
 26. The method of any one of claims 1-25, wherein saidadministering results in a therapeutic effect.
 27. The method of claim26, wherein the targeting moiety is capable of binding to atumor-associated antigen, and said therapeutic effect comprises anincrease in T cells specific for the tumor-associated antigen.
 28. Themethod of claim 27, wherein said increase in T cells occurs in thetumor.
 29. The method of claim 27 or 28, wherein said increase in the Tcells in the tumor is relative to T cells in the spleen.
 30. The methodof claim 27, 28, or 29, wherein said administering results in at least15% of the total T cell population in a sample from the mammal beingspecific for the tumor-associated antigen.
 31. The method of claim 30,wherein said administering results in at least 20% of the total T cellpopulation in a sample from the mammal being specific for thetumor-associated antigen.
 32. The method of claim 31, wherein saidadministering results in at least 25% of the total T cell population ina sample from the mammal being specific for the tumor-associatedantigen.
 33. The method of claim 32, wherein said administering resultsin at least 30% of the total T cell population in a sample from themammal being specific for the tumor-associated antigen.
 34. The methodof claim 33, wherein said administering results in at least 35% of thetotal T cell population in a sample from the mammal being specific forthe tumor-associated antigen.
 35. The method of claim 34, wherein saidadministering results in at least 40% of the total T cell population ina sample from the mammal being specific for the tumor-associatedantigen.
 36. The method of claim 35, wherein said administering resultsin at least 45% of the total T cell population in a sample from themammal being specific for the tumor-associated antigen.
 37. The methodof claim 36, wherein said administering results in at least 50% of thetotal T cell population in a sample from the mammal being specific forthe tumor-associated antigen.
 38. The method of claim 37, wherein saidadministering results in at least 55% of the total T cell population ina sample from the mammal being specific for the tumor-associatedantigen.
 39. The method of claim 38, wherein said administering resultsin at least 60% of the total T cell population in a sample from themammal being specific for the tumor-associated antigen.
 40. The methodof claim 40, wherein said administering results in at least 65% of thetotal T cell population in a sample from the mammal being specific forthe tumor-associated antigen.
 41. The method of claim 41, wherein saidadministering results in at least 70% of the total T cell population ina sample from the mammal being specific for the tumor-associatedantigen.
 42. The method of any one of claims 30-41, wherein the sampleis a tumor sample.
 43. The method of any one of claims 26-42, whereinsaid therapeutic effect comprises a decrease in tumor volume, a stabletumor volume, or a reduced rate of increase in tumor volume.
 44. Themethod of any one of claims 26-43, wherein said therapeutic effectcomprises a decreased incidence of recurrence or metastasis.